In vitro evolution of uropathogenic Escherichia coli to fosfomycin resistance in a 3D cultured human bladder microtissue model

In vitro studies of antimicrobial resistance (AMR) using laboratory growth media produce important, fundamental information. However, their inability to more closely replicate the in vivo environment limits the translational potential of this work. Here, we used a 3D cultured microtissue model which reflects the human bladder microenvironment to select for resistance to fosfomycin in two uropathogenic strains of Escherichia coli, UTI-34 and UTI-59. To assess the clinical relevance of the mutations produced, we screened the observed mutations in the fosfomycin-selected variants against a curated dataset of 14,163 E. coli genomes isolated from urine. The four independent fosfomycin-selected variants of UTI-34 contained diverse mutations, while the mutations in the five independent fosfomycin-selected variants of UTI-59 were more constrained. All variants contained mutations in glpT, uhpT, uhpA and uhpC, which are commonly linked to fosfomycin-resistance in clinical isolates of E. coli. Screening of the mutations against the 14,163 E. coli genomes from urine confirmed that four of these mutations were found as exact matches in the dataset, while other mutation types were confirmed at a regional and gene level. These mutations did not result in any collateral susceptibility or resistance to other antibiotics recommended for the treatment of urinary tract infections. The use of a human 3D microtissue model, which closely replicates the urothelial microenvironment to study AMR during urinary tract infection treatment, could improve the clinical relevance of in vitro AMR studies. This has the potential to provide a better understanding of how AMR is acquired and expressed, and inform new strategies to combat AMR.