Running title: Plasmid-Dependent Phages in Wastewater Microbiome
Conjugative plasmids are major drivers of horizontal gene transfer and antibiotic resistance (AMR) dissemination in wastewater. Plasmid-dependent phages (PDPs) infect bacteria carrying conjugative plasmids by targeting plasmid-encoded mating structures, offering a potential strategy to limit plasmid persistence. Here, we evaluated the impact of three PDPs (PRD1, Lu221, and Hgal1) on domestic and hospital wastewater microbiomes using serial cultivation and metagenomic sequencing at 24 h and 48 h. In the absence of phages, communities converged toward Enterobacterales-dominated assemblages, with reduced diversity and enrichment of ESBL-producing bacteria and AMR-carrying conjugative plasmids. Phage addition led to replication of most introduced and resident phages, except PRD1, which declined over time. Despite active infection, PDPs had minimal effects on total bacterial abundance, taxonomic composition, or phenotypic resistance. However, metagenomic analyses revealed source- and treatment-specific shifts in conjugative plasmid types, particularly within MPF_F and MPF_T systems. PRD1 induced transient increases in conjugative plasmids, while resident phages promoted enrichment of MPF_F in the hospital community. These effects were restricted to plasmid-level dynamics and did not scale to detectable community-level changes. Overall, PDPs exert selective pressure on plasmid populations but remain decoupled from broader community outcomes under high-turnover conditions.
Conjugative plasmids are major drivers of horizontal gene transfer and antibiotic resistance (AMR) dissemination in wastewater. Plasmid-dependent phages (PDPs) infect bacteria carrying conjugative plasmids by targeting plasmid-encoded mating structures, offering a potential strategy to limit plasmid persistence. Here, we evaluated the impact of three PDPs (PRD1, Lu221, and Hgal1) on domestic and hospital wastewater microbiomes using serial cultivation and metagenomic sequencing at 24 h and 48 h. In the absence of phages, communities converged toward Enterobacterales-dominated assemblages, with reduced diversity and enrichment of ESBL-producing bacteria and AMR-carrying conjugative plasmids. Phage addition led to replication of most introduced and resident phages, except PRD1, which declined over time. Despite active infection, PDPs had minimal effects on total bacterial abundance, taxonomic composition, or phenotypic resistance. However, metagenomic analyses revealed source- and treatment-specific shifts in conjugative plasmid types, particularly within MPF_F and MPF_T systems. PRD1 induced transient increases in conjugative plasmids, while resident phages promoted enrichment of MPF_F in the hospital community. These effects were restricted to plasmid-level dynamics and did not scale to detectable community-level changes. Overall, PDPs exert selective pressure on plasmid populations but remain decoupled from broader community outcomes under high-turnover conditions.
DTU Bioengineering