Development of a novel streamlined workflow (AACRE) and database (inCREDBle) for genomic analysis of carbapenem-resistant Enterobacterales.

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In response to the threat of increasing antimicrobial resistance, we must increase the amount of available high-quality genomic data gathered on antibiotic-resistant bacteria. To this end, we developed an integrated pipeline for high-throughput long-read sequencing, assembly, annotation and analysis of bacterial isolates and used it to generate a large genomic data set of carbapenemase-producing Enterobacterales (CPE) isolates collected in Spain. The set of 461 isolates were sequenced with a combination of both Illumina and Oxford Nanopore Technologies (ONT) DNA sequencing technologies in order to provide genomic context for chromosomal loci and, most importantly, structural resolution of plasmids, important determinants for transmission of antimicrobial resistance. We developed an informatics pipeline called Assembly and Annotation of Carbapenem-Resistant Enterobacteriaceae (AACRE) for the full assembly and annotation of the bacterial genomes and their complement of plasmids. To explore the resulting genomic data set, we developed a new database called inCREDBle that not only stores the genomic data, but provides unique ways to filter and compare data, enabling comparative genomic analyses at the level of chromosomes, plasmids and individual genes. We identified a new sequence type, ST5000, and discovered a genomic locus unique to ST15 that may be linked to its increased spread in the population. In addition to our major objective of generating a large regional data set, we took the opportunity to compare the effects of sample quality and sequencing methods, including R9 versus R10 nanopore chemistry, on genome assembly and annotation quality. We conclude that converting short-read and hybrid microbial sequencing and assembly workflows to the latest nanopore chemistry will further reduce processing time and cost, truly enabling the routine monitoring of resistance transmission patterns at the resolution of complete chromosomes and plasmids.