Bacterial Concentrations and Water Turbulence Influence the Importance of Conjugation Versus Phage-Mediated Antibiotic Resistance Gene Transfer in Suspended Growth Systems

[Image: see text] Despite the abundance of phage-borne antibiotic resistance genes (ARGs) in the environment, the frequency of ARG propagation via phage-mediated transduction (relative to via conjugation) is poorly understood. We investigated the influence of bacterial concentration and water turbulence level [quantified as Reynold’s number (Re)] in suspended growth systems on the frequency of ARG transfer by two mechanisms: delivery by a lysogenic phage (phage λ carrying gentamycin-resistance gene, genR) and conjugation mediated by the self-transmissible plasmid RP4. Using Escherichia coli (E. coli) as the recipient, phage delivery had a comparable frequency (1.2 ± 0.9 × 10(–6)) to that of conjugation (1.1 ± 0.9 × 10(–6)) in suspensions with low cell concentration (10(4) CFU/mL) and moderate turbulence (Re = 5 × 10(4)). Turbulence affected cell (or phage)-to-cell contact rates and detachment (due to shear force), and thus, it affected the relative importance of conjugation versus phage delivery. At 10(7) CFU/mL, no significant difference was observed between the frequencies of ARG transfer by the two mechanisms under quiescent water conditions (2.8 ± 0.3 × 10(–5) for conjugation vs 2.2 ± 0.5 × 10(–5) for phage delivery, p = 0.19) or when Re reached 5 × 10(5) (3.4 ± 1.5 × 10(–5) for conjugation vs 2.9 ± 1.0 × 10(–5) for phage delivery, p = 0.52). Transcriptomic analysis of genes related to conjugation and phage delivery and simulation of cell (or phage)-to-cell collisions at different Re values corroborate that the importance of phage delivery relative to conjugation increases under either quiescent or turbulent conditions. This finding challenges the prevailing view that conjugation is the dominant ARG transfer mechanism and underscores the need to consider and mitigate potential ARG dissemination via transduction.