Extended-spectrum cephalosporin-resistant Escherichia coli (ESC-R- Ec ) is an urgent public health threat with sequence type clonal complex 131 (STc131), phylogroup B2 strains being particularly concerning as the dominant cause of ESC-R- Ec infections. To address the paucity of recent ESC-R- Ec molecular epidemiology data in the United States, we used whole-genome sequencing (WGS) to fully characterize a large cohort of invasive ESC-R- Ec at a tertiary care cancer center in Houston, Texas, collected from 2016 to 2020. During the study time frame, there were 1,154 index E. coli bloodstream infections (BSIs) of which 389 (33.7%) were ESC-R- Ec . Using time series analyses, we identified a temporal dynamic of ESC-R- Ec distinct from ESC-susceptible E. coli (ESC-S- Ec ), with cases peaking in the last 6 months of the calendar year. WGS of 297 ESC-R- Ec strains revealed that while STc131 strains accounted for ~45% of total BSIs, the proportion of STc131 strains remained stable across the study time frame with infection peaks driven by genetically heterogeneous ESC-R- Ec clonal complexes. bla CTX-M variants accounted for most β-lactamases conferring the ESC-R phenotype (89%; 220/248 index ESC-R -Ec ), and amplification of bla CTX-M genes was widely detected in ESC-R- Ec strains, particularly in carbapenem non-susceptible, recurrent BSI strains. Bla CTX-M-55 was significantly enriched within phylogroup A strains, and we identified bla CTX-M-55 plasmid-to-chromosome transmission occurring across non-B2 strains. Our data provide important information regarding the current molecular epidemiology of invasive ESC-R- Ec infections at a large tertiary care cancer center and provide novel insights into the genetic basis of observed temporal variability for these clinically important pathogens. IMPORTANCE Given that E. coli is the leading cause of worldwide ESC-R Enterobacterales infections, we sought to assess the current molecular epidemiology of ESC-R- Ec using a WGS analysis of many BSIs over a 5-year period. We identified fluctuating temporal dynamics of ESC-R- Ec infections, which have also recently been identified in other geographical regions such as Israel. Our WGS data allowed us to visualize the stable nature of STc131 over the study period and demonstrate a limited but genetically diverse group of ESC-R- Ec clonal complexes are detected during infection peaks. Additionally, we provide a widespread assessment of β-lactamase gene copy number in ESC-R- Ec infections and delineate mechanisms by which such amplifications are achieved in a diverse array of ESC-R- Ec strains. These data suggest that serious ESC-R- Ec infections are driven by a diverse array of strains in our cohort and impacted by environmental factors suggesting that community-based monitoring could inform novel preventative measures.

Extended-spectrum cephalosporin resistant (ESC-R) Escherichia coli (ESC-R-Ec) is an urgent public health threat with clonal complex (CC) 131, phylogroup B2 strains being particularly concerning as the dominant cause of ESC-R-Ec infections. To address the paucity of recent ESC-R-Ec molecular epidemiology data in the United States (US), we used whole genome sequencing (WGS) to fully characterize a large cohort of invasive ESC-R-Ec at a tertiary care cancer center in Houston, Texas collected from 2016-2020. During the study timeframe, there were 1154 index E. coli bloodstream infections (BSIs) of which 389 (33.7%) were ESC-R. Using time series analyses, we identified a temporal dynamic of ESC-R E. coli BSIs (Ec-BSIs), distinct from ESC-susceptible Ec-BSIs, with cases peaking in the last 6 months of the calendar year. WGS of 297 ESC-R Ec-BSI strains revealed that while CC131 strains accounted for ~45% of total infections, the proportion of CC131 strains remained stable across the time-period, and infection peaks were driven by genetically diverse, non-CC131 isolates. BlaCTX-M variants accounted for most beta-lactamases conferring the ESC-R phenotype (89%; 220/248 index ESC-R Ec-BSIs), and amplification of blaCTX-M genes was widely detected in ESC-R Ec-BSI strains, particularly in carbapenem non-susceptible strains and in strains causing recurrent BSIs. BlaCTX-M-55 was significantly enriched within phylogroup A strains, and we identified blaCTX-M-55 plasmid-to-chromosome transmission occurring across non-B2 strains. Our data provide important information regarding the current molecular epidemiology of invasive ESC-R E. coli and provide novel insights into the genetic basis of observed temporal variability for these clinically important pathogens.

Currently, whole genome sequencing (WGS) data has not shown strong concordance with E. 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 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 blaTEM 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 blaTEM in ESRI, 112/134 strains (84%) with at least ampicillin non-susceptibility encoded blaTEM. Evidence of blaTEM amplification (i.e., blaTEM gene copy number estimates > 2x;) was present in 40/112 (36%) strains. There were positive correlations between blaTEM 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 beta-lactam resistance mechanisms, including non-ceftriaxone hydrolyzing blaCTX-M variants, blaOXA-1, as well as ampC and blaTEM 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 beta-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 beta-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 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.