Genomic surveillance of multidrug-resistant organisms based on long-read sequencing
Authors: Landman Fabian , Jamin Casper , de Haan Angela , Witteveen Sandra , Bos Jeroen , van der Heide Han G.J. , Schouls Leo M. , Hendrickx Antoni P.A. ,   Dutch CPE and MRSA surveillance study groups
Published: December 2024

BackgroundMultidrug-resistant organisms (MDRO) pose a significant threat to public-health world-wide. The ability to identify antimicrobial resistance determinants, to assess changes in molecular types, and to detect transmission are essential for effective 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 rapidly and accurately characterize MDRO for surveillance and transmission studies based on long-read sequencing only.MethodsGenomic DNA of 356 MDRO was automatically extracted using the Maxwell-RSC48. The MDRO included 106Klebsiella pneumoniaeisolates, 85Escherichia coli, 15Enterobacter cloacaecomplex, 10Citrobacter freundii, 34Pseudomonas aeruginosa, 16Acinetobacter baumannii, and 69 methicillin-resistantStaphylococcus 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 >40x coverage was used for multi-locus sequence typing (MLST), whole-genome MLST (wgMLST),in silicomultiple locus variable-number of tandem repeat analysis (iMLVA) for MRSA, and identification of resistance genes (Abricate).ResultsComparison 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 forP. aeruginosa.The wgMLST profiles obtained by long-read and short-read WGS differed only one to nine wgMLST alleles forK. pneumoniae,E. coli,E. cloacaecomplex,C. freundii,A. baumanniicomplex and MRSA. ForP. aeruginosadifferences were up to 27 wgMLST alleles between long-read and short-read wgMLST. MLST sequence types andin silicoMLVA 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.ConclusionsWe demonstrate that molecular characterization of automatically extracted DNA followed by long-read sequencing is as accurate and more cost-effective compared to short-read sequencing. Long-read sequencing is suitable for molecular typing and outbreak analysis as part of genomic surveillance of MDRO. However, the analysis ofP. aeruginosarequires further improvement which may be obtained by other basecalling algorithms. The low implementation costs, low price per isolate, and rapid library preparation for long-read sequencing of MDRO extends its applicability to resource-constrained settings and low-income countries world-wide.