Systematic Analysis of Mobile Genetic Elements Mediating β-Lactamase Gene Amplification in Noncarbapenemase-Producing Carbapenem-Resistant Bloodstream Infections.

Noncarbapenemase-producing carbapenem-resistant (non-CP-CRE) are increasingly recognized as important contributors to prevalent carbapenem-resistant (CRE) infections. However, there is limited understanding of mechanisms underlying non-CP-CRE causing invasive disease. Long- and short-read whole-genome sequencing was used to elucidate carbapenem nonsusceptibility determinants in bloodstream isolates at MD Anderson Cancer Center in Houston, Texas. We investigated carbapenem nonsusceptible (CNSE) mechanisms (i.e., isolates with carbapenem intermediate resistance phenotypes or greater) through a combination of phylogenetic analysis, antimicrobial resistance gene detection/copy number quantification, porin assessment, and mobile genetic element (MGE) characterization. Most CNSE isolates sequenced were non-CP-CRE (41/79; 51.9%), whereas 25.3% (20/79) were with intermediate susceptibility to carbapenems (CIE), and 22.8% (18/79) were carbapenemase-producing (CPE). Statistically significant copy number variants (CNVs) of extended-spectrum β-lactamase (ESBL) genes (Wilcoxon Test; -value < 0.001) were present in both non-CP-CR E. coli (median CNV = 2.6×; = 17) and K. pneumoniae (median CNV = 3.2×,  = 17). All non-CP-CR E. coli and K. pneumoniae had predicted reduced expression of at least one outer membrane porin gene (i.e., or ). Completely resolved CNSE genomes revealed that IS and IS structures harboring variants along with other antimicrobial resistance elements were associated with gene amplification, occurring in mostly IncFIB/IncFII plasmid contexts. MGE-mediated β-lactamase gene amplifications resulted in either tandem arrays, primarily mediated by IS translocatable units, or segmental duplication, typically due to IS transposition units. Non-CP-CRE strains were the most common cause of CRE bacteremia with carbapenem nonsusceptibility driven by concurrent porin loss and MGE-mediated amplification of genes. Carbapenem-resistant (CRE) are considered urgent antimicrobial resistance (AMR) threats. The vast majority of CRE research has focused on carbapenemase-producing (CPE) even though noncarbapenemase-producing CRE (non-CP-CRE) comprise 50% or more of isolates in some surveillance studies. Thus, carbapenem resistance mechanisms in non-CP-CRE remain poorly characterized. To address this problem, we applied a combination of short- and long-read sequencing technologies to a cohort of CRE bacteremia isolates and used these data to unravel complex mobile genetic element structures mediating β-lactamase gene amplification. By generating complete genomes of 65 carbapenem nonsusceptible (CNSE) covering a genetically diverse array of isolates, our findings both generate novel insights into how non-CP-CRE overcome carbapenem treatments and provide researchers scaffolds for characterization of their own non-CP-CRE isolates. Improved recognition of mechanisms driving development of non-CP-CRE could assist with design and implementation of future strategies to mitigate the impact of these increasingly recognized AMR pathogens.