Brown, C. S. | Search bioRxiv

bioRxiv2025

Nanopore long-read only genome assembly of clinical Enterobacterales isolates is complete and accurate

Whole bacterial genome sequence reconstruction using Oxford Nanopore Technologies ("Nanopore") long-read only sequencing may offer a lower-cost, higher-throughput alternative for pathogen surveillance to hybrid assembly with recent improvements in Nanopore sequencing accuracy. We evaluated the accuracy, including plasmid reconstruction, of Nanopore long-read only genome assemblies of Enterobacterales. We sequenced 92 genomes from clinical Enterobacterales isolates, collected in England under a national surveillance program, with long-read Nanopore (R10.4.1, Dorado v5.0.0 super-high-accuracy basecalled) and short-read Illumina (NovaSeq) sequencing approaches. Genomes were assembled using three long-read only (Flye; Hybracter long; Autocycler), and three hybrid assemblers (Hybracter hybrid; Unicycler normal; bold). Three polishing modalities (Medaka v2 with subsampled or un-subsampled long-reads; Polypolish + Pypolca with short-reads) were investigated. Autocycler circularised the most chromosomes (87/92 [95%]). Plasmid sequence reconstruction was comparable between all assemblers except Flye, all recovering 90-96% of plasmids, although the ground truth was uncertain. Flye performed worse than other assemblers on almost all metrics. Autocycler + Medaka (un-subsampled long-reads) was the most accurate long-read only assembler/polisher combination, comparable to hybrid assemblies (median 0 [IQR:0-0] SNPs and 0 [IQR:0-1] indels per genome; quality value/Q score, 100 [IQR: 64-100]), with only 4/92 genome sequences having >10 SNPs/indels. Medaka polishing with un-subsampled long-reads resulted in small improvements in indels but not SNPs for both Flye and Autocycler assemblies. Seven-locus MLST, antimicrobial resistance, virulence, and stress gene annotation was equivalent across assembler/polisher combinations. Nanopore long-read only bacterial genome assembly with Autocycler combined with Medaka polishing (using un-subsampled reads) is similarly accurate and possibly more complete than hybrid assemblies, representing a viable alternative for incorporating high-quality genomic data, including plasmids, into Enterobacterales surveillance. Data SummaryNanopore long-reads and Illumina short-reads from the 92 Enterobacterales isolates from this study have been uploaded to ENA (BioProject accession: PRJEB93885). Code for the Nextflow assembly pipeline, downstream analysis scripts, and R statistical analysis scripts are available on GitHub (https://github.com/oxfordmmm/NEKSUS_ont_hybrid_assembly_comparison). The following supplementary data tables are available on FigShare (https://figshare.com/account/home#/projects/253775): O_LIENA Sample accessions and sample metadata (accessions_and_metadata.csv) C_LIO_LISeqkit stats summaries of the Illumina and Nanopore reads (raw_qc_sup.cav) C_LIO_LISummary of assembly contig features (contigs_summary_sup_cleaned.csv) C_LIO_LIPairwise mash distances between contigs (mash_cleaned.csv) C_LIO_LIPlasmids matching across different assemblers compared to the Hybracter (hybrid) and manually-curated reference sets (plasmids_match_hybracter_mash.csv; plasmids_match_manual_mash.csv, respectively) C_LIO_LISeven-locus multi-locus sequence type annotation (mlst_cleaned.csv) C_LIO_LICheckM2 summaries of assemblies (checkm2_cleaned.csv) C_LIO_LINucleotide-level accuracy of assemblies (SNP, Indels, and Quality value compared to short-read mapping; assembly_nucleotide_accuracy_cleaned.csv) C_LIO_LIBakta annotation (bakta_by_contig_cleaned.csv) C_LIO_LIAMRFinderPlus annotations of contigs (amrfinder_plus_cleaned.csv) C_LIO_LIMOB-suite annotation summaries of contigs (mobsuite_cleaned.csv) C_LI Impact StatementNanopore long-reads have historically been too error-prone to use alone for accurate bacterial genome assembly, necessitating additional Illumina short-reads to achieve structurally complete and accurate hybrid genome assemblies for public health surveillance. This increases cost and complexity. Previous studies have shown that recent improvements in Nanopore chemistry (R10.4.1 flowcell) and basecalling (super-high accuracy) allow high-quality long-read only assemblies on a small number of laboratory reference strains. This is the first evaluation, to our knowledge, to assess Nanopore long-read only genome assembly compared with hybrid assembly on a large number of clinical isolates. In addition, this is the first large-scale evaluation of the recently released automated consensus long-read assembly tool, Autocycler. We show that Autocycler long-read only assemblies are more structurally complete for chromosomal sequences, while reconstructing a similar number of plasmids to other long-read and hybrid assemblers. Most long-read polished, Autocycler-assembled genome sequences have 0 errors (median: 0 SNPs/indels) relative to a short-read polished (hybrid) Autocycler assemblies, enabling accurate annotation of key genes.

bioinformatics↗

bioRxiv2023

IS1-related large-scale deletion of chromosomal regions harbouring oxygen-insensitive nitroreductase gene nfsB causes nitrofurantoin heteroresistance in Escherichia coli

Nitrofurantoin is a broad-spectrum first-line antimicrobial used for managing uncomplicated urinary tract infection. Loss-of-function mutations in chromosomal genes nfsA, nfsB, and ribE of Escherichia coli are known to reduce nitrofurantoin susceptibility. Here, we report monoclonal nitrofurantoin heteroresistance in E. coli and a novel genetic mechanism associated with this phenomenon. Subpopulations with reduced nitrofurantoin susceptibility in cultures of two E. coli blood strains were identified using population analysis profiling. Four colonies of each strain growing on agar with 0.5xMIC nitrofurantoin were sub-cultured in broth with 0.5xMIC nitrofurantoin (n=2) or without nitrofurantoin (n=2). Moreover, one colony of each strain growing without nitrofurantoin exposure was selected as a reference for genomic comparison. Whole-genome sequencing of all isolates were conducted on Illumina and Nanopore MinION systems. Both strains had a nitrofurantoin MICs of 64 mg/L. The proportion of cells grown at 0.5xMIC was two and 99 per million, respectively, which is distinct to that of a homogeneously susceptible or resistant isolate. All isolates grown at 0.5xMIC had 11-66 kbp deletions in chromosomal regions harbouring nfsB, and all these deletions were immediately adjacent to IS1-family insertion sequences. Although this study is limited to E. coli and nitrofurantoin, our findings suggest IS1-associated genetic deletion represents a hitherto unrecognised mechanism of heteroresistance that could compromise infection management and impact conventional antimicrobial susceptibility testing. Impact statementNitrofurantoin is widely used for treating and preventing urinary tract infection. Prevalence of nitrofurantoin resistance generally is low in E. coli. Here, we report nitrofurantoin heteroresistance in two E. coli blood strains and attribute this phenotype to IS1-associated deletion of chromosomal regions harbouring oxygen-insensitive nitroreductase gene nfsB. Our discoveries demonstrate a novel genetic mechanism of heteroresistance and suggest detecting nitrofurantoin heteroresistance in E. coli urinary isolates for improving prescribing. Data summaryWhole-genome sequencing reads and genome assemblies generated in this study have been deposited under BioProject PRJEB58678 in the European Nucleotide Archive (ENA). Accession numbers are listed in Supplementary Table 1. Previously generated Illumina whole-genome sequencing reads of parental isolates EC0026B and EC0880B are available under ENA accessions ERR3142418 and ERR3142524, respectively.

microbiology↗

bioRxiv2017

Multiuse patient monitoring equipment as a risk factor for acquisition of Candida auris

Introduction Introduction Methods Results Discussion References Candida auris is an emerging and multi-drug resistant pathogen recently associated with outbreaks worldwide, often in intensive care settings (1). Following the alerts issued by the CDC and PHE in June 2016 (2,3), a look back exercise in the neurological intensive care unit (NITU) in the Oxford University Hospitals NHS Foundation Trust identified five clinical cases of C. auris infection. After the identification of an additional isolate in a urine sample, a patient and environmental screening programme was introduced in the last week of October 2016. We report here a summary of our initial findings which other affected units may wish to consider as part of their infection prevention and control measures. ...

microbiology↗