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

Abstract

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 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.

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.

Keywords: ESBL gene amplification; experimental evolution; non-carbapenemase carbapenem resistance; pseudo compound transposon; sequence type 131.

Figure 1.

Population structure of sequence type (ST) 131 Escherichia coli bacteremia isolates. Midpoint rooted, recombination-masked, core genome–inferred maximum-likelihood phylogeny of ST131 E coli (n = 184) collected from 2013 to 2020 at our institution. The 3 major subclones with clade C split into C1 and C2 are labeled on the dendrogram. Terminal branch tips are colored by subcladal structure (n = 11) as identified using a Bayesian analysis of population structure. Columnar metadata are labeled with corresponding categories presented in the figure key at right. Thirteen ST131 isolates from 9 subclades that were selected for carbapenem mutation frequency analysis are labeled by stars that appear to the right of columnar metadata. Abbreviations: AST, antimicrobial susceptibility testing; CNV, copy number variant; CPE, carbapenemase-producing Enterobacterales; ESBL, extended-spectrum β-lactamase; ESC-R, extended-spectrum cephalosporin resistant; ESC-S, extended-spectrum cephalosporin susceptible; non-CP-CRE, non-carbapanemase-producing carbapenem resistant Enterobacterales.

Figure 2.

Sequence type (ST) 131 fluctuation assay mutant genomic characterization. Ertapenem (ETP) minimum inhibitory concentration (MIC) fold change (×) (A) and extended-spectrum β-lactamase (ESBL) gene fold change (×) (B) across 5 predominant subclades of ST131. The y-axis in each respective subpanel is presented in log₁₀ scale without data transformation. Variant status is labeled by color as presented in the figure key. ESBL copy number variant (CNV) between parental (ie, carbapenem naive) and progeny mutant (ie, positive growth following ETP exposure) strain stratified by isolates with no variant of interest (C) versus strains with a variant of interest detected (D), with clade indicated by color in the key. Dunn multiple-comparisons test: **P < .01; *P < .05.

Figure 3.

Growth of MB1860 with increasing ertapenem (ETP) concentration gradients. A, Flask transfer protocol (FTP) populations (n = 3) exposed to stepwise gradient increase in ETP concentrations with indication of positive growth (optical density at 600 nm [OD₆₀₀] >0.5) at each respective day. B, Note that population 1–3 stepwise growth followed comparable trajectories up to day 9 of passaging microfluidic system (MFS) populations (n = 3) with OD₆₀₀ measurements indicated on the left y-axis and ETP concentration stepwise increments (gray solid line) indicated on the right y-axis. Dotted lines indicate variance in OD₆₀₀ measurements over time, with each population color-coded in the key. A fitted loess curve (solid color-coded lines) for OD₆₀₀ measurements of MFS populations is juxtaposed across the line graph. Blue horizontal dotted line indicates ancestral MB1860 ETP minimum inhibitory concentration (0.0625 μg/mL). Red horizontal dotted line (A) indicates Clinical and Laboratory Standards Institute carbapenem resistance breakpoint [19]. Red arrows indicate whole genome sequencing sampling timepoints.

Figure 4.

Amplification of the MB1860 pseudocompound transposon (PCTn) and Western blot analysis of outer membrane porins (OMPs) in flask transfer protocol (FTP) and microfluidic system (MFS) experiments. FTP (A) and MFS (B) normalized coverage depth of MB1860 PCTns indicating amplification of β-lactamases bla_OXA-1 (blue open reading frame) and bla_CTX-M-15 (green open reading frame) as presented on the x-axis. Red arrows indicate where nonsense mutations were detected corresponding with each particular day. FTP and MFS population 3 (P3) isolates are illustrated here as representatives of their respective evolutionary trajectories. A large-scale figure of the MB1860 PCTn shown in relation to the ompC gene is presented in Supplementary Figure 2. FTP (C) and MFS (D) immunoblot analysis of OmpC across select daily population and end point isolates (EPIs). “OmpC^– control” is the previously characterized OmpC-negative strain MB9877 [5] and “OmpC⁺ control” is the positive control, parental MB1860 strain. Red and blue boxes correspond to red and blue arrows in (A) and (B), respectively, highlighting strains of interest.

Figure 5.

Transcriptome analysis of MB1860 daily population isolates in flask transfer protocol (FTP) and microfluidic system (MFS) platforms. Differential expression analysis was conducted on daily population isolates (FTP population 1, day 2 isolates and MFS population 1, day 26 isolates) and exposed to 0.5× ertapenem (ETP) minimum inhibitory concentration (ie, 0.03 µg/mL ETP) compared to passage controls without ETP exposure. Volcano plots of differentially expressed genes in the FTP (A) and MFS (B) platforms with genes of interest identified. Dotted lines demarcate genes that have an adjusted P value cutoff of 1e-6 and log₂ fold change = 2, with red dots indicating statistically significant results with absolute log₂ fold change (FC) values >2. Gene set enrichment analysis of FTP (C) and MFS (D) platforms with activated vs suppressed gene sets. Gene ratio indicates total fraction of genes belonging to that particular gene set with statistically upregulated or downregulated expression and count labeled in the key, respectively.