Genomic Surveillance of Multidrug-Resistant Organisms Based on Long-Read Sequencing

Research Background

Multidrug-resistant organisms (MDROs) are a significant global public health threat. These organisms exhibit resistance to multiple antibiotics, making infections challenging to treat and increasing healthcare costs. To effectively monitor and control the spread of MDROs, accurate identification of antimicrobial resistance genes, changes in molecular types, and transmission pathways is crucial. Traditional molecular typing methods, such as pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST), have played an essential role in the past but are limited by low resolution, complexity, and high costs. In recent years, whole-genome sequencing (WGS)-based methods have become mainstream, with short-read sequencing technologies (e.g., Illumina platforms) widely used in genomic surveillance. However, short-read sequencing has limitations in detecting structural variations such as large insertions, deletions, and repetitive sequences, which hinders complete bacterial genome assembly.

Long-read sequencing technologies, such as Oxford Nanopore Technologies (ONT), have emerged as promising alternatives due to their ability to generate long DNA fragments. These technologies improve genome assembly completeness and provide more accurate detection of resistance genes and plasmid structures. This study aims to evaluate the accuracy and applicability of long-read sequencing for genomic surveillance of MDROs in molecular typing and transmission analysis.

Paper Source

This paper, authored by Fabian Landman, Casper Jamin, and others from the National Institute for Public Health and the Environment (RIVM) in the Netherlands, was published in Genome Medicine in 2024. The title of the paper is Genomic Surveillance of Multidrug-Resistant Organisms Based on Long-Read Sequencing.

Research Workflow

1. Sample Selection and DNA Extraction

The study included 356 MDRO isolates, comprising 106 Klebsiella pneumoniae, 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 isolates were associated with an outbreak. Genomic DNA was extracted using the Maxwell® RSC48 automated extraction platform.

2. Sequencing and Data Analysis

Samples underwent both short-read (Illumina NextSeq 550) and long-read (Nanopore Rapid Barcoding Kit-24-v14, R10.4.1) WGS. Long-read data were basecalled using Dorado-0.3.2 and assembled with various tools, including Flye, Canu, Miniasm, Unicycler, NECAT, Raven, and Redbean. Short-read data were assembled using the Juno-assembly v2.0.2 pipeline.

3. Molecular Typing and Resistance Gene Identification

Long-read and short-read sequencing results were compared using MLST, whole-genome MLST (wgMLST), and whole-genome single-nucleotide polymorphisms (wgSNP). Resistance genes and plasmid replicons were identified using the ResFinder and PlasmidFinder databases.

Key Findings

1. Comparison of Long-Read and Short-Read Sequencing

Long-read sequencing results for wgMLST were highly concordant with short-read data for most MDROs, with differences ranging from 1 to 9 alleles. However, for Pseudomonas aeruginosa, the difference was more pronounced, with up to 27 alleles. MLST and MLVA profiles from long-read and short-read sequencing were entirely consistent.

2. Antimicrobial Resistance Gene Identification

Long-read sequencing demonstrated high sensitivity and specificity (92–100%/99–100%) in antimicrobial resistance (AMR) gene detection. It also provided advantages in identifying multicopy resistance genes and plasmid structures, which are critical for understanding AMR mechanisms and outbreaks.

3. MRSA Outbreak Analysis

The study successfully analyzed an MRSA outbreak in the central Netherlands using long-read sequencing. The wgMLST, MLST, and MLVA profiles from long-read sequencing were highly consistent with short-read results, demonstrating the accuracy of long-read sequencing in outbreak investigations.

Conclusion

The study demonstrates that long-read sequencing is a reliable method for genomic surveillance of MDROs, offering comparable accuracy to short-read sequencing in molecular typing, AMR gene detection, and outbreak analysis. Long-read sequencing also provides complete genome assemblies and improved detection of structural variations and multicopy resistance genes. Additionally, the relatively low cost and rapid library preparation make it a practical solution for resource-limited settings worldwide.

Research Highlights

1. High Accuracy: Long-read sequencing matched short-read results for most MDROs, confirming its reliability in genomic surveillance.
2. Resistance Gene Detection: Long-read sequencing accurately identified AMR genes, including multicopy genes on mobile genetic elements, which were challenging for short-read sequencing.
3. Outbreak Analysis: Long-read sequencing effectively analyzed the MRSA outbreak, underscoring its potential in epidemiological investigations.
4. Low Cost and Rapid Preparation: Long-read sequencing’s cost-effectiveness and quicker preparation make it suitable for low-resource settings.

Implications

This study offers a cost-effective and efficient long-read sequencing method for genomic surveillance of MDROs. It improves genome assembly completeness and resilience when analyzing complex structures like resistance plasmids. The findings provide critical support for MDRO prevention and control strategies, emphasizing the potential for leveraging long-read sequencing in global MDRO surveillance programs, especially in resource-constrained environments.