Genetic determinants underlying the progressive phenotype of β-lactam/β-lactamase inhibitor resistance in
Escherichia coli
Authors: Shropshire William C. , Amiji Hatim , Bremer Jordan , Selvaraj Anand Selvalakshmi , Strope Benjamin , Sahasrabhojane Pranoti , Gohel Marc , Aitken Samuel , Spitznogle Sarah , Zhan Xiaowei , Kim Jiwoong , Greenberg David E. , Shelburne Samuel A. 
Published: October 2023

Currently, whole-genome sequencing (WGS) data have not shown strong concordance with
Escherichia coli
susceptibility profiles to the commonly used β-lactam/β-lactamase inhibitor (BL/BLI) combinations: ampicillin-sulbactam (SAM), amoxicillin-clavulanate (AMC), and piperacillin-tazobactam (TZP). Progressive resistance to these BL/BLIs in the absence of cephalosporin resistance, also known as extended-spectrum resistance to BL/BLI (ESRI), has been suggested to primarily result from increased copy numbers of
bla
TEM
variants, which is not routinely assessed in WGS data. We sought to determine whether addition of gene amplification could improve genotype-phenotype associations through WGS analysis of 147
E. coli
bacteremia isolates with increasing categories of BL/BLI non-susceptibility ranging from ampicillin (AMP) susceptibility to being fully resistant to all three BL/BLIs. Consistent with a key role of
bla
TEM
in ESRI, 112/134 strains (84%) with at least ampicillin non-susceptibility encoded
bla
TEM
. Evidence of
bla
TEM
amplification (i.e.,
bla
TEM
gene copy number estimates > 2×) was present in 40/112 (36%) strains. There were positive correlations between
bla
TEM
copy numbers with minimum inhibitory concentrations of AMC and TZP (
P
< 0.05) but not for SAM (
P
= 0.09). The diversity of β-lactam resistance mechanisms, including non-ceftriaxone hydrolyzing
bla
CTX-M
variants,
bla
OXA-1
, and
ampC
and
bla
TEM
strong promoter mutations, was greater in AMC- and TZP-non-susceptible strains but rarely observed within SAM- and AMP-non-susceptible isolates. Our study indicates that comprehensive analysis of WGS data, including β-lactamase-encoding gene amplification, can help categorize
E. coli
with AMC or TZP non-susceptibility but that discerning the transition from SAM susceptibility to SAM non-susceptibility using genetic data requires further refinement.

IMPORTANCE

The increased feasibility of whole-genome sequencing has generated significant interest in using such molecular diagnostic approaches to characterize difficult-to-treat, antimicrobial-resistant (AMR) infections. Nevertheless, there are current limitations in the accurate prediction of AMR phenotypes based on existing AMR gene database approaches, which primarily correlate a phenotype with the presence/absence of a single AMR gene. Our study utilized a large cohort of cephalosporin-susceptible
Escherichia coli
bacteremia samples to determine how increasing the dosage of narrow-spectrum β-lactamase-encoding genes in conjunction with other diverse β-lactam/β-lactamase inhibitor (BL/BLI) genetic determinants contributes to progressively more severe BL/BLI phenotypes. We were able to characterize the complexity of the genetic mechanisms underlying progressive BL/BLI resistance including the critical role of β-lactamase encoding gene amplification. For the diverse array of AMR phenotypes with complex mechanisms involving multiple genomic factors, our study provides an example of how composite risk scores may improve understanding of AMR genotype/phenotype correlations.