Journal Article

Genomic surveillance of multidrug-resistant organisms based on long-read sequencing

Schouls, Leo M., Witteveen, Sandra, Jamin, Casper, Hendrickx, Antoni P. A., Bos, Jeroen, de Haan, Angela, Landman, Fabian, van der Heide, Han G. J.

2024

Overview

Background Multidrug-resistant organisms (MDRO) pose a significant threat to public health worldwide. The ability to identify antimicrobial resistance determinants, to assess changes in molecular types, and to detect transmission are essential for surveillance and infection prevention of MDRO. Molecular characterization based on long-read sequencing has emerged as a promising alternative to short-read sequencing. The aim of this study was to characterize MDRO for surveillance and transmission studies based on long-read sequencing only. Methods Genomic DNA of 356 MDRO was automatically extracted using the Maxwell-RSC48. The MDRO included 106 Klebsiella pneumoniae isolates, 85 Escherichia coli , 15 Enterobacter cloacae complex, 10 Citrobacter freundii , 34 Pseudomonas aeruginosa , 16 Acinetobacter baumannii , and 69 methicillin-resistant Staphylococcus aureus (MRSA), of which 24 were from an outbreak. MDRO were sequenced using both short-read (Illumina NextSeq 550) and long-read (Nanopore Rapid Barcoding Kit-24-V14, R10.4.1) whole-genome sequencing (WGS). Basecalling was performed for two distinct models using Dorado-0.3.2 duplex mode. Long-read data was assembled using Flye, Canu, Miniasm, Unicycler, Necat, Raven, and Redbean assemblers. Long-read WGS data with > 40 × coverage was used for multi-locus sequence typing (MLST), whole-genome MLST (wgMLST), whole-genome single-nucleotide polymorphisms (wgSNP), in silico multiple locus variable-number of tandem repeat analysis (iMLVA) for MRSA, and identification of resistance genes (ABRicate). Results Comparison of wgMLST profiles based on long-read and short-read WGS data revealed > 95% of wgMLST profiles within the species-specific cluster cut-off, except for P. aeruginosa . The wgMLST profiles obtained by long-read and short-read WGS differed only one to nine wgMLST alleles or SNPs for K. pneumoniae , E. coli , E. cloacae complex, C. freundii , A. baumannii complex, and MRSA. For P. aeruginosa , differences were up to 27 wgMLST alleles between long-read and short-read wgMLST and 0–10 SNPs. MLST sequence types and iMLVA types were concordant between long-read and short-read WGS data and conventional MLVA typing. Antimicrobial resistance genes were detected in long-read sequencing data with high sensitivity/specificity (92–100%/99–100%). Long-read sequencing enabled analysis of an MRSA outbreak. Conclusions We demonstrate that molecular characterization of automatically extracted DNA followed by long-read sequencing is as accurate compared to short-read sequencing and suitable for typing and outbreak analysis as part of genomic surveillance of MDRO. However, the analysis of P. aeruginosa requires further improvement which may be obtained by other basecalling algorithms. The low implementation costs and rapid library preparation for long-read sequencing of MDRO extends its applicability to resource-constrained settings and low-income countries worldwide.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Alleles / Analysis / Anopheles / Antimicrobial resistance / Automation / Bacteria - classification / Bacteria - drug effects / Bacteria - genetics / Bacterial pneumonia / Bioinformatics / Biomedical and Life Sciences / Biomedicine / Cancer Research / CPE / CPPA / CRAB / DNA methylation / DNA sequencing / Drug resistance / Drug resistance in microorganisms / Drug Resistance, Multiple, Bacterial - genetics / Escherichia coli / Genes / Genome, Bacterial / Genomes / Genomic analysis / Genomic surveillance / Genomics / Genomics - methods / Health aspects / High-Throughput Nucleotide Sequencing - methods / Human Genetics / Humans / Klebsiella pneumoniae / Laboratories / Long-read sequencing / Medicine/Public Health / Metabolomics / Methicillin / MRSA / Multidrug resistance / Multidrug resistant organisms / Multilocus sequence typing / Nucleotide sequence / Nucleotide sequencing / Outbreaks / Plasmids / Pneumonia / Pseudomonas aeruginosa / Public health / Single nucleotide polymorphisms / Single-nucleotide polymorphism / Staphylococcus infections / Surveillance / Systems Biology / Whole genome sequencing / Whole Genome Sequencing - methods