Genetic determinants underlying the progressive phenotype of β-lactam/β-lactamase inhibitor resistance in Escherichia coli

Currently, whole-genome sequencing (WGS) data have not shown strong concordance with Escherichia coli susceptibility profiles to the commonly used beta-lactam/beta-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 > 2x) 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 beta-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 beta-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 beta-lactamase-encoding genes in conjunction with other diverse beta-lactam/beta-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 beta-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.