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

Antimicrobial resistance is recognized as a major global health threat. Pathogens can rapidly evolve resistance which diminishes the impact of antimicrobial treatments. The presence of other microbes in polymicrobial infections can impact the development of antimicrobial resistance of pathogens, yet it is unclear how the interplay between the environmental structure and the presence of a polymicrobial community affect the rate of plasmid mediated antibiotic resistance evolution. This study investigates the effect of environmental structure and microbial interactions on antibiotic resistance evolution in a uropathogenic Escherichia coli. We conducted a serial transfer experiment across well-mixed and spatially structured environ-ments with increasing concentrations of trimethoprim-sulfamethoxazole, 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 spatial structures allowed for prolonged resistance evolution. Copy number variation of the plasmid-borne resistance locus varied significantly across the different evolutionary contexts. E. coli lineages evolved in the absence of the community, in the well-mixed isolation environ-ments demonstrated the highest copy number increases, while those in the presence of the community showed minimal changes compared to the ancestor. These findings underscore the intricate relationship between the genetic basis of resistance, the environmental structure and the microbial ecology in plasmid-mediated antimicrobial resistance evolution.