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

## TL;DR

This study explores how a high-risk strain of Escherichia coli adapts to carbapenem antibiotics, revealing that gene amplification is a key initial step toward resistance.

## Contribution

The study identifies IS26-mediated gene amplification as a conserved initial adaptation mechanism in ESBL-positive ST131 E. coli under carbapenem exposure.

## Key findings

- Initial carbapenem exposure leads to ESBL gene amplification via IS26-mediated transposons in ST131 C2/H30Rx isolates.
- Increased ESBL gene copy number correlates strongly with higher mutation frequencies in ESBL-positive ST131 strains.
- Stable porin gene mutations occur only after prolonged carbapenem exposure, suggesting a sequential adaptation process.

## Abstract

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.

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.

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 blaCTX-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.

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.

High-risk ST131 Escherichia coli strains, particularly C2/H30Rx subclades, adapt to initial carbapenem exposure by insertion sequence–mediated extended-spectrum β-lactamase gene amplification. This gene amplification may precede stable mutations in porin genes, potentially leading to resistance with manageable fitness costs.