Comprehensive Assessment of Initial Adaptation of Extended-Spectrum β-Lactamase–Positive ST131 Escherichia coli to Carbapenem Exposure

Background. It remains unclear how high-risk Escherichia coli lineages, like sequence type (ST) 131, initially adapt to carbapenem exposure in their progression to carbapenem resistance. Methods. Carbapenem mutation frequency was measured in multiple subclades of extended-spectrum β-lactamase (ESBL)–positive ST131 clinical isolates using a fluctuation assay followed by whole genome sequencing (WGS) characterization. Genomic, transcriptomic, and porin analyses of the ST131 C2/H30Rx isolate MB1860, under prolonged, increasing carbapenem exposure was performed using 2 experimental evolutionary platforms to measure fast versus slow adaptation. Results. All 13 ESBL-positive ST131 strains selected from a diverse (n = 184) ST131 bacteremia cohort had detectable ertapenem (ETP) mutational frequencies, with a positive correlation between initial ESBL gene copy number and mutation frequency (r = 0.87, P < 1e-5). WGS analysis of mutants showed that initial response to ETP exposure resulted in significant increases in ESBL gene copy numbers or mutations in Omp genes in the absence of ESBL gene amplification with subclade-specific associations. In both experimental evolutionary platforms, MB1860 responded to initial ETP exposure by increasing bla_CTX-M-15 copy numbers via modular, IS26-mediated pseudocompound transposons (PCTns). Increased transcript level of genes present within the PCTn was a conserved expression signal in both experimental evolutionary platforms. Stable mutations in Omp encoding genes were detected only after prolonged increasing carbapenem exposure, consistent with clinical observations. Conclusions. ESBL gene amplification is a conserved response to initial carbapenem exposure, especially within the high-risk ST131 C2/H30Rx subclade. Targeting such amplification could assist with mitigating carbapenem resistance development.