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

Abstract

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.

Keywords: antimicrobial resistance; carbapenem-resistant Enterobacterales; database; genome assembly; nanopore sequencing.

Fig. 1.

Distribution of (a) species, (b) communities and (c) species by geographical location on a map of Spain, and sequence types for (d) K. pneumoniae , (e) E. cloacae and (f) E. coli .

Fig. 2.

The AACRE workflow. Pre-processing steps are shown in green. Pre-assembly quality control steps to verify the purity and identity of the samples are shown in red. Assembly by Unicycler is shown in purple. Annotation steps are shown in orange.

Fig. 3.

Phylogenetic relationship among isolates. Phylogenetic trees of core genomes for (a) E. coli , (b) E. cloacae complex and (c) K. pneumoniae were annotated with multilocus sequence type (ST), carbapenemases present (OXA-48, KPC-2/KPC-3, VIM-1, NDM or IMP), the community in Spain where strains were isolated and the collection from which they came (SERGAS or CNM). The E. cloacae complex includes the following species: Enterobacter hormaechei subsp . hoffmannii (ST78), Enterobacter xiangfangensis (ST171, ST114, ST1379, ST66, ST182), Enterobacter roggenkampii (ST96, ST515) and Enterobacter hormaechei subsp. steigerwaltii (ST93, ST133) [56, 57].

Fig. 4.

Clustering of plasmid sequences by average nucleotide identity. Plasmid sequences defined as sequences with length <1 Mbp identified by PlasmidFinder were clustered using the similarity metric tANI. Plasmid types (replicons and MOB types) are labelled in (a, c), the carbapenemase gene that they carry in (d), the community where they were isolated in (b), and the species and sequence type in (e, f) (zoomed in). The plasmid similarity network shown here can be explored interactively at https://resistome.cnag.cat/incredble/ani/, where these images were generated.

Fig. 5.

Representative ST15-unique gene cluster in the assembled genome of sample AH0330, an OXA-48-positive K. pneumoniae isolate collected at the Hospital Universitario Central de Asturias (Oviedo, Spain). Sample details at https://genomes.cnag.cat/incredble/277/.