Anaerobic conditions increase plasmid transfer rates across Escherichia coli strains

Bacterial conjugation is the primary mechanism by which antibiotic resistance genes spread in microbial populations, yet our understanding of this process has been largely based on experiments conducted under aerobic conditions. This creates a fundamental disconnect: environments that are considered hotspots for gene exchange (e.g., the gut, abscesses, chronic wounds, and wastewater systems) are predominantly anaerobic. In this study, we investigate whether oxygen availability influences the transfer rate of a set of common ESBL-IncI1- and qnrS1-IncF plasmids in commensal Escherichia coli strains from chickens. We found that oxygen availability significantly shapes conjugation dynamics in a recipient strain-specific manner, with anaerobic conditions promoting higher ESBL-IncI1- plasmid transfer rates to commensal E. coli recipients. Conjugation rates of the ESBL-IncI1- plasmids to a laboratory strain of E. coli were several orders of magnitude higher and independent of oxygen level, while two qnrS1-IncF plasmids showed higher anaerobic rates. Our study reveals critical "oxygen blind spots" in conventional conjugation assays and suggests that conventional aerobic conjugation assays underestimate plasmid transfer rates in natural environments such as the chicken caeca. These findings highlight the importance of aligning experimental conditions with the physiological and ecological environments in which gene exchange naturally occurs. Tailoring these variables is essential for generating results that accurately reflect, predict, and potentially intervene in the horizontal spread of antimicrobial resistance.