When things add up: environmental structure and microbial interactions drive antibiotic-resistance plasmid evolution

Background and objectives Antimicrobial resistance is a major global health threat, driven in part by the rapid evolution of resistance in pathogens, which undermines the effectiveness of antimicrobial treatment. In infections, pathogens rarely live in a well-mixed, single species environment. It is an open question how microbial interactions in contrasting environmental structures affect plasmid mediated antibiotic resistance evolution. Methodology This study investigates how a spatially structured versus a well-mixed liquid environment, together with microbial interactions, affect antibiotic resistance evolution in uropathogenic Escherichia coli. We conducted a serial transfer experiment under increasing concentrations of trimethoprim-sulfamethoxazole comparing resistance evolution in the well-mixed and spatially structured environments, both in the presence and absence of a polymicrobial community.

Our results revealed that E. coli in community context displayed parallel evolutionary trajectories, leading to higher final antibiotic tolerance, while the spatial structure allowed for prolonged resistance evolution. Copy number variation of the plasmid-borne resistance locus varied significantly across conditions; E. coli evolved in the well-mixed, monoculture conditions, exhibited the greatest increases in copy number, whereas lineages evolved in the presence of the community showed minimal changes relative to the ancestor.

and implications These findings underscore the complex interplay between the genetic basis of resistance, the environmental structure and microbial ecology in shaping plasmid-mediated antimicrobial resistance evolution. Lay summary Antibiotic resistance depends on environmental context, microbial interactions, and genetics. This study shows that E. coli evolved resistance differently in mixed versus structured environments, with changes in plasmid-borne resistance gene copy number. Community interactions led to similar evolutionary paths and higher tolerance, while well-mixed, single-species conditions drove larger genetic changes. Competing Interest Statement The authors have declared no competing interest. Footnotes The manuscript has been shortened, and readability has been improved.