As, C. G. | bioRxiv Science

bioRxiv2026

Assessment of Oxford Nanopore whole genome sequencing for large-scale genomic characterisation of Staphylococcus aureus

Whole-genome sequencing (WGS) is increasingly being utilised in microbial diagnostics, surveillance, and research. In this paper we assess the performance of one leading long-read sequencing technology, Oxford Nanopore Technology (ONT), on 836 Staphylococcus aureus bacteraemia isolates. We compare the results to that of a leading short-read sequencing technology, Illumina. All isolates were sequenced using ONT MinION Mk1B and Illumina HiSeq or MiSeq. Libraries were prepared according to manufacturers instructions. Preprocessing and downstream bioinformatic analyses were performed using a combination of in-house pipelines and publicly available software tools. The average base substitution error rate in ONT assemblies was low but varied between sequence types, possibly due to lineage-specific methylation patterns. Multi locus sequence typing was similar between the technologies, while ONT assemblies allowed for better spa typing than Illumina assemblies. The reported detection rate was similar between ONT and Illumina assemblies for most virulence- and AMR-associated genes and variants. For 42 (22.2%) of 189 genes/variants, the two technologies disagreed in gene detection in 5 isolates or more, and in 39 (20.6.%) of these the highest detection rate was found with ONT. Discrepancies were mainly associated with low GC content, multiple repetitive segments, and small plasmids. Polishing of ONT data resulted in minor changes in gene/variant calling. Our study supports the use of ONT WGS for bacterial population genomic studies on a large collection of S. aureus isolates. While assembly of ONT reads may be affected by its own methodological limitations, it was superior to Illumina assemblies in detection of potentially clinically relevant genes and variants at a low read error rate. Understanding the advantages and limitations of WGS technologies is essential before undertaking studies involving such methods on large sets of bacteria. Author summaryIn this paper, we present a practical assessment of one important whole genome sequencing (WGS) method, Oxford Nanopore Technology (ONT), and compare its performance in bacterial population genomics to that of WGS with Illumina technology. Our goal was to investigate the usefulness of ONT in studies aiming to identify clinically relevant bacterial characteristics in large collections of bacteria, such as genotype-phenotype studies. We sequenced a large set of clinical S. aureus isolates from episodes of bloodstream infections using both ONT and Illumina technologies and performed analyses with widely used software and bioinformatic pipelines. We have elucidated inherent strengths and limitations of ONT and Illumina sequencing and report some of the practical consequences of these on bacterial typing and detection of clinically relevant genes. With this study, we present one of the most comprehensive assessments of long-read sequencing technology for the genomic characterisation of clinical bacterial isolates, and the findings provide guidance for researchers considering WGS in large-scale bacterial genomics.

genomics↗

bioRxiv2025

Trends and characteristics of multidrug resistant MRSA in Norway 2008-2020

Infections caused by multidrug-resistant (MDR) bacteria are recognized as a critical One Health concern which poses a significant threat to public health, leading to increased morbidity and mortality across both high- and low-income countries. In this study, we investigated the epidemiology and molecular mechanisms of multidrug-resistant methicillin-resistant Staphylococcus aureus (MDR-MRSA) strains identified in Norway from 2008 to 2020, in order to gain a better understanding of the evolution and dissemination of multidrug resistance in S. aureus. A total of 452 MDR-MRSA strains isolated from 429 individuals were analyzed from a dataset of 23,412 MRSA strains. Methods included epidemiological characterization, antimicrobial susceptibility testing (AST) and genetic analysis of a selection of strains using nanopore sequencing to identify antimicrobial resistance (AMR) genes and mutations, as well as their location on plasmids, SCCmec and other mobile genetic elements (MGEs). The study revealed an overall increasing trend in MDR-MRSA strains, with healthcare-associated strains being more prevalent among MDR-MRSA compared to the overall MRSA population. Significant heterogeneity in spa-types and clonal complexes exhibiting multidrug resistance was observed, with high resistance rates against multiple antibiotic groups, particularly erythromycin, ciprofloxacin/norfloxacin, tetracycline, gentamicin, and clindamycin in addition to cefoxitin. The predominant MDR-MRSA clones included t1476/CC8, t127/CC1, t189/CC188 and t030, t037/CC239. A broad range of AMR genes and mutations were detected, linked to a wide variety of MGEs, highlighting the complex mechanisms of resistance development and dissemination within the MRSA population. This study highlights the rising challenge posed by MDR-MRSA strains, and reveals the multifactorial nature of AMR in S. aureus, thus emphasizing the importance of continued surveillance, antibiotic stewardship and infection control measures, as well as global cooperation, in order to combat the spread of these multidrug-resistant pathogens. Author SummaryIn our study, we explored the landscape of multidrug-resistant methicillin-resistant Staphylococcus aureus (MDR-MRSA) in Norway from 2008 to 2020. This research is possible because it draws on a robust national surveillance system that has been active for over a decade, aimed at preventing the establishment of these dangerous pathogens in our healthcare facilities. While the overall incidence of MDR-MRSA was relatively low, we noticed an upward trend in the number of these resistant strains over time. This pattern, along with shifts in the molecular profiles of the strains, suggests that certain MDR-MRSA clones have become well-established and are spreading globally. One of the most important findings was that the majority of MDR-MRSA strains were acquired abroad. This indicates that international travel and migration are significant contributors to the spread of these resistant strains, particularly from regions like Asia and Africa. This underscores the necessity for global collaboration in surveillance and antibiotic stewardship to combat the threat posed by these pathogens. Additionally, we found that a high proportion of MDR-MRSA strains were associated with healthcare settings, primarily isolated from patients during hospital admissions. This is concerning, as it suggests that the most resistant strains are often found in hospitals, where vulnerable patients are at risk. The high antibiotic exposures in these environments likely contributes to the selection and spread of these resistant clones. Interestingly, we discovered that many of the MDR-MRSA strains were detected in asymptomatic carriers rather than in clinical infections. This could be due to the strains being acquired abroad and subsequently identified through routine screening in healthcare settings. The overall potential implications for public health are however significant, especially since the resistance profiles of these strains can severely limit treatment options. By utilizing advanced nanopore sequencing technology, we were able to delve deeper into the genetic elements responsible for antibiotic resistance, highlighting the extensive heterogeneity of resistance mechanisms among the MDR-MRSA strains. We found that resistance genes are primarily located on plasmids and other mobile genetic elements, which enhances their potential for spread among different strains. This complexity of resistance mechanisms and the adaptive strategies employed by MRSA highlight the ongoing battle against antibiotic resistance. In conclusion, our study sheds light on the evolving landscape of MDR-MRSA, emphasizing the need for continued vigilance and coordinated efforts to mitigate the spread of these resistant strains.

microbiology↗