Tracing the evolutionary dynamics of carbapenem-resistant Escherichia coli in recurrent and multi-site infections.

BACKGROUND: Carbapenem-resistant Escherichia coli (CREC) can cause persistent or multi-site infections, leading to significant clinical challenges due to the limited availability of effective antibiotics. However, the within-host evolution of CREC and its impact on infection patterns remain poorly understood. This study aims to characterize CREC isolates from patients with recurrent or multi-site infections to elucidate the relationship between bacterial adaptation within the host and infection dynamics, thereby addressing a critical gap in our understanding of CREC pathogenesis. RESULTS: Genotypic analysis, including Nanopore whole-genome sequencing, and phenotypic comparisons were performed on CREC isolates from individual patients. Pulsed-field gel electrophoresis (PFGE) patterns revealed that 18 patients were consistently infected with highly genetically related strains. Moreover, two patients (Patients 16 and 18) experienced sequential infections caused by genetically distinct strains, resulting in a total of 20 strain groups. Among these, seven (35%) belonged to phylogroup B1, six (30%) to phylogroup A, four (20%) to phylogroup B2, and three (15%) to phylogroup D. Nine groups were multidrug-resistant (MDR), six were extensively drug-resistant (XDR), and four shifted from XDR to MDR. Notably, group 18 - 1 included two MDR and five XDR strains. We examined the distribution of 31 virulence-associated genes across 20 groups and found that only three groups carried less than 10 genes. However, all strains within the same group harbored the same set of virulence genes. Larvae infection models revealed that strains from patients 7 and 8 became increasingly virulent over time, while those from patients 11 and 16 showed reduced virulence. Plaque assays revealed variability in phage susceptibility among isolates from different patients, as well as among consecutive isolates obtained from the same patient over time. Whole-genome sequencing results suggested plasmid dissemination among CREC strains in patients 5 and 18 based on highly identical plasmid sequences. CONCLUSIONS: These findings underscore the significance of bacterial genomic changes and plasmid transfer in driving phenotypic evolution, enabling CREC to adapt and persist within hosts under selective pressures, thereby sustaining infections.