Systematic Analysis of Mobile Genetic Elements Mediating β-Lactamase Gene Amplification in Noncarbapenemase-Producing Carbapenem-Resistant <i>Enterobacterales</i> Bloodstream Infections

Abstract

Noncarbapenemase-producing carbapenem-resistant <i>Enterobacterales</i> (non-CP-CRE) are increasingly recognized as important contributors to prevalent carbapenem-resistant <i>Enterobacterales</i> (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 <i>Enterobacterales</i> bloodstream isolates at MD Anderson Cancer Center in Houston, Texas. We investigated carbapenem nonsusceptible <i>Enterobacterales</i> (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 <i>Enterobacterales</i> with intermediate susceptibility to carbapenems (CIE), and 22.8% (18/79) were carbapenemase-producing <i>Enterobacterales</i> (CPE). Statistically significant copy number variants (CNVs) of extended-spectrum β-lactamase (ESBL) genes (Wilcoxon Test; <i>P</i>-value < 0.001) were present in both non-CP-CR E. coli (median CNV = 2.6×; <i>n</i> = 17) and K. pneumoniae (median CNV = 3.2×, <i>n</i> = 17). All non-CP-CR E. coli and K. pneumoniae had predicted reduced expression of at least one outer membrane porin gene (i.e., <i>ompC/ompF</i> or <i>ompK36/ompK35</i>). Completely resolved CNSE genomes revealed that IS<i>26</i> and IS<i>Ecp1</i> structures harboring <i>bla</i>_CTX-M 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<i>26</i> translocatable units, or segmental duplication, typically due to IS<i>Ecp1</i> 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 <i>bla</i>_CTX-M genes. <b>IMPORTANCE</b> Carbapenem-resistant <i>Enterobacterales</i> (CRE) are considered urgent antimicrobial resistance (AMR) threats. The vast majority of CRE research has focused on carbapenemase-producing <i>Enterobacterales</i> (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 <i>Enterobacterales</i> (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.