IS26-mediated amplification of blaOXA-1 and blaCTX-M-15 with concurrent outer membrane porin disruption associated with de novo carbapenem resistance in a recurrent bacteraemia cohort

Abstract

Background Approximately half of clinical carbapenem-resistant Enterobacterales (CRE) isolates lack carbapenem-hydrolysing enzymes and develop carbapenem resistance through alternative mechanisms. Objectives To elucidate development of carbapenem resistance mechanisms from clonal, recurrent ESBL-positive Enterobacterales (ESBL-E) bacteraemia isolates in a vulnerable patient population. Methods This study investigated a cohort of ESBL-E bacteraemia cases in Houston, TX, USA. Oxford Nanopore Technologies long-read and Illumina short-read sequencing data were used for comparative genomic analysis. Serial passaging experiments were performed on a set of clinical ST131 Escherichia coli isolates to recapitulate in vivo observations. Quantitative PCR (qPCR) and qRT–PCR were used to determine copy number and transcript levels of β-lactamase genes, respectively. Results Non-carbapenemase-producing CRE (non-CP-CRE) clinical isolates emerged from an ESBL-E background through a concurrence of primarily IS26-mediated amplifications of blaOXA-1 and blaCTX-M-1 group genes coupled with porin inactivation. The discrete, modular translocatable units (TUs) that carried and amplified β-lactamase genes mobilized intracellularly from a chromosomal, IS26-bound transposon and inserted within porin genes, thereby increasing β-lactamase gene copy number and inactivating porins concurrently. The carbapenem resistance phenotype and TU-mediated β-lactamase gene amplification were recapitulated by passaging a clinical ESBL-E isolate in the presence of ertapenem. Clinical non-CP-CRE isolates had stable carbapenem resistance phenotypes in the absence of ertapenem exposure. Conclusions These data demonstrate IS26-mediated mechanisms underlying β-lactamase gene amplification with concurrent outer membrane porin disruption driving emergence of clinical non-CP-CRE. Furthermore, these amplifications were stable in the absence of antimicrobial pressure. Long-read sequencing can be utilized to identify unique mobile genetic element mechanisms that drive antimicrobial resistance.

Accessory Genomes Drive Independent Spread of Carbapenem-Resistant Klebsiella pneumoniae Clonal Groups 258 and 307 in Houston, TX

“…All isolates identified as CR Kp in our sampling frame were subjected to Oxford Nanopore Technologies (ONT) long-read sequencing using the SQK-RBK004 library preparation kit and sequenced on an Oxford Nanopore GridION X5 (Oxford, UK). The hybrid assembly pipeline has been described previously ( 38 ). Genome assembly metrics are shown in Table S4 (at https://gitlab.com/carmig_dissertations/shropshire_dissertation/crkp_supplemental_files ) with their respective BioSample accession and Antibacterial Resistance Leadership Group (ARLG) identification numbers.…”

Systematic Analysis of Mobile Genetic Elements Mediating β-lactamase Gene Amplification in Non-Carbapenemase-Producing Carbapenem Resistant Enterobacterales Bloodstream Infections

“…Copy number variant profiling of β-lactamases encoding genes in CNSE An increase in copy number of ESBL, AmpC-like, and narrow-spectrum β-lactamase encoding genes has been previously documented as contributing to CNSE development (13,23,25,26).…”

“…A primary factor responsible for the dissemination of MDR phenotypes are mobile genetic elements (MGEs). These complex genetic structures (e.g., plasmids, transposons, integrons) can mobilize carbapenem resistance determinants in addition to other antimicrobial resistance (AMR) genes that confer resistance to other classes of antibiotics such as fluoroquinolones, aminoglycosides, and other novel β-lactam/β-lactamase inhibitor combinations (9)(10)(11)(12)(13). In recent years, the development of long-read sequencing technologies has improved our understanding of the complexity, diversity, and prevalence of these MGEs as key drivers of MDR infections (13)(14)(15)(16)(17)(18).…”

“…These studies have shown that non-CP-CRE development typically involves increased expression or gene copy number of ESBL or AmpClike enzymes in conjunction with outer membrane porin (omp) gene inactivation, which results in a reduced carbapenem concentration in the periplasmic space (22)(23)(24)(25)(26). Given that both ESBL and AmpC-like encoding genes are typically located in MGEs (11,13,27), an increase in βlactamase gene copy number would seem to be feasible for a broad array of ESBL and AmpClike positive Enterobacterales. To our knowledge, a systematic analysis of MGE mediated βlactamase encoding gene amplifications in a large cohort of CRE isolates using completed genome assemblies has not been performed.…”

Candida aurisInvasive Infections during a COVID-19 Case Surge

“…Illumina-based assemblies were generated as previously described ( 4 ), and the hybrid-assembly of NC_1 was generated using a bespoke pipeline ( https://github.com/wshropshire/flye_hybrid_assembly_pipeline ). A core gene phylogenetic tree using a representative set of reference genomes was generated to assess phylogenetic clustering and confirm species identification, as previously described ( 5 ). Single nucleotide polymorphisms (SNPs) were identified with GATK v4.1.9.0 ( 6 ) using the best practices workflow and NC_1 as an internal reference.…”