Genetic Determinants Underlying the Progressive Phenotype of β-lactam/β-lactamase Inhibitor Resistance inEscherichia coli

Currently, whole genome sequencing (WGS) data has not shown strong concordance with E. 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 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-susceptible to 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 (MICs) of AMC and TZP ( P -value < 0.05), but not for SAM ( P -value = 0.09). The diversity of β-lactam resistance mechanisms, including non-ceftriaxone hydrolyzing bla CTX-M variants, bla OXA-1 , as well as ampC and bla TEM strong promoter mutations, were greater in AMC and TZP non-susceptible strains but rarely observed within SAM and AMP non-susceptible isolates. Our study indicates a 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 susceptible to non-susceptible 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 E. coli bacteremia samples to determine how increasing dosage of narrow-spectrum β-lactamase encoding genes in conjunction with other diverse BL/BLI genetic determinants contribute 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.