Genes on the Move: Exploring Microbiomes within Aquatic Ecosystems of First Nation Communities in Manitoba

Oxidation lagoons (OLs) are widely used wastewater treatment systems in rural and First Nation (FN) communities in Canada and are essential for protecting public health. Although designed to remove pollutants and pathogens, OLs may facilitate the exchange of mobile genetic elements and antibiotic resistance genes (ARGs), promoting antimicrobial resistance (AMR) among environmental bacteria and bacteriophages. This study investigated AMR dynamics by characterizing the resistome of bacterial isolates and metagenomic samples from an OL serving an FN community in Manitoba.

We combined wet- and dry-lab approaches to examine viable bacteria and bacterial and phage metagenomes across treatment stages. Thirty-five samples were collected between September 2022 and April 2023 from raw sewage (RS), lagoon (LG), a submerged attached growth reactor (SAGR), UV-disinfected effluent (EF), and an upstream control site (UPS) including surface water and sediment. Genomic and metagenomic DNA were sequenced using Oxford Nanopore Technology with de novo assembly. Viable isolates were enumerated, and bacterial and Escherichia coli counts were quantified using digital PCR.

Among 58 isolates, Aeromonas (50.87%) and Serratia (15.78%) predominated, with a lower representation of Pantoea, Escherichia, Lelliottia, Rahnella, Enterobacter, Buttiauxella, Acinetobacter, Yersinia, and Citrobacter. ARG analysis identified 32,559 ARG-associated elements, including 425 strict or perfect hits in 84.5% of isolates. Genes conferring resistance to carbapenems, β-lactams, macrolides, tetracyclines, and fluoroquinolones were detected. An Acinetobacter radioresistens isolate from UV-treated effluent carried blaOXA-23 and showed resistance to imipenem (64 µg/mL). Metagenomics (84 samples) revealed Caudoviricetes as the dominant viral class, with aminoglycoside, fluoroquinolone, and phenicol resistance genes most abundant. Gene copy numbers were highest at UPS and RS (1.48 and 1.10 × 10⁵ cells/mL). E. coli averaged 172.5 cells/mL in RS.

These findings demonstrate the persistence of clinically relevant bacteria and ARGs in treated wastewater, supporting the need for AMR surveillance in decentralized systems serving FN communities.