Biology subjects

Sauerborn, E.

Publications and source records attributed to Sauerborn, E..

4 recordsLinked to original sources

bioRxiv2026

Verification of nanopore sequencing technology for clinical carbapenem-resistant Enterobacterales surveillance

Carbapenem-resistant Enterobacterales (CRE) pose a critical threat to global public health and often contribute to the rapid plasmid-mediated dissemination of carbapenemase genes. While established routine diagnostics can confirm the presence of the most common carbapenemases, these approaches do not resolve the genomic context of resistance and thus cannot confirm transmission events, cross-species dissemination, or atypical resistance mechanisms. Nanopore sequencing-based whole-genome sequencing (WGS) can capture this genomic context through complete de novo genome and plasmid assemblies. However, for routine clinical use of nanopore WGS for CRE surveillance, direct comparisons with established diagnostics and clear guidelines on required sequencing depths are needed. We used 100 carbapenemase-producing CRE isolates from routine diagnostics at a tertiary-care hospital to compare results from WGS against established diagnostics, and determined the sequencing depth required for species identification, strain typing, carbapenemase detection, and plasmid-level epidemiology. We additionally examined 10 carbapenem-non-susceptible CRE isolates, for which routine diagnostics identified no carbapenemase gene despite phenotypic carbapenem non-susceptibility. Across all isolates, nanopore WGS reproduced routine carbapenemase family and pathogen detections, and additionally resolved the carbapenemase subtypes and their genomic context, the bacterial species and strain, and resistance mechanisms that established diagnostics had missed. Such strain typing and plasmid-level resolution are essential for infection control responses to differentiate between clonal spread of CRE, dissemination of shared plasmid, or unrelated infection events. Our study thus strongly supports the integration of cost-efficient nanopore WGS into CRE diagnostics, surveillance, and outbreak investigation. The required sequencing depth depends on the clinical objective, with species identification being reliable at a depth of 10x, strain typing and carbapenemase detection at a depth of at least 20x, and robust plasmid-level characterisation at a depth of at least 40x. Across our CRE collection, the detected carbapenemases were mostly plasmid-borne, and predominantly encoded by relatively conserved IncN and more heterogenous IncL/M plasmids. ImportanceCarbapenem-resistant bacteria are among the most serious threats in modern medicine, leaving clinicians with few treatment options. Nanopore sequencing can be a powerful tool to rapidly and precisely track resistance and guide infection control, but limited comparisons with clinically established diagnostics and uncertainty about how much sequencing data is needed currently limit routine clinical use. We show that nanopore sequencing detects all relevant carbapenemase genes identified by routine diagnostics, resolves carbapenem resistance mechanisms that standard tests miss, and generally increases the resolution of pathogen characterizations for transmission and outbreak tracing. We provide guidance on the sequencing depth required for diagnostic tasks, from identifying species to tracking plasmid-borne resistance genes across time and pathogens. By benchmarking nanopore sequencing against established diagnostics and matching sequencing effort to the clinical question, we offer a framework that makes genomic surveillance of carbapenem-resistant bacteria accessible and cost-efficient.

genomics↗

bioRxiv2026

Machine learning-guided discovery of a conserved plasmid proteomic signature enables MALDI-TOF MS detection of pOXA-48-carrying Enterobacterales

OXA-48 carbapenemases are among the most widespread and important resistance mechanisms in Enterobacterales. Yet detecting carbapenemases by conventional workflows necessitates additional testing, thus delaying optimization of therapy and implementation of infection control measures. Here, we present a machine learning approach that identifies the conserved pOXA-48 plasmid directly from routine MALDI-TOF spectra acquired for species identification. The model detects pOXA-48 carriers with an AUROC of 0.96-0.98 across two independent hospital cohorts and instrument platforms, indicating near-perfect discrimination. Using bottom-up proteomics, plasmid conjugation, and plasmid curing, we link the discriminative MALDI-TOF spectral features to proteins encoded on pOXA-48, with DUF1496 domain-containing protein producing the most discriminative spectral feature. Our approach reframes the resistance prediction task from inferring a resistance phenotype to detecting a conserved plasmid through its expressed proteomic signature and has the potential to enable rapid MALDI-TOF MS-based diagnostics for a wide range of plasmid-based resistance determinants.

microbiology↗

bioRxiv2026

Nanopore metagenomic sequencing links clinically relevant resistance determinants to pathogens

Culture-independent metagenomics enables the detection of plasmid-encoded antimicrobial resistance (AMR) genes directly from clinical samples; however, the clinical significance of these genes depends on their bacterial host and genomic context, which metagenomics cannot fully infer. Nanopore sequencing technology intrinsically encodes epigenetic modifications such as methylation, which can be leveraged for plasmid-host associations from metagenomic data. Existing methods rely on the recovery of metagenome-assembled genomes (MAGs), which can introduce bias toward abundant taxa and leave clinically relevant, low-abundance pathogens unassociated. To address this limitation, we extended methylation-based plasmid-host association from the MAG level to individual assembly contigs and sequencing reads. The CUPID pipeline implements the calculation of contig and read similarity scores, which compare weighted mean methylation rates across motifs genetically shared between any contig or read pair. We validated this approach on a mock metagenomic community composed of ten carbapenem-resistant Enterobacterales isolates, where we achieved 93.8% accuracy at the contig level and 100% at the read level for carbapenemase plasmid-host associations. When applied to metagenomic and quasimetagenomic data of sixteen patient rectal swabs collected during routine hospital surveillance, our approach assigned every detected plasmid-encoded carbapenemase to its correct bacterial host at the contig level, using matched culture-based diagnostics and whole-genome sequencing as a ground truth. Read-level analysis identified additional associations that were missed at the contig level, including a multi-host plasmid confirmed by established diagnostics. These findings demonstrate a pathway from rapid AMR gene detection using metagenomics to actionable surveillance for infection prevention, transmission tracing, and outbreak investigation. Impact statementCulture-independent metagenomics can detect antimicrobial resistance genes, but their clinical significance depends on the bacterial host and genomic context. Here, we show that nanopore-derived bacterial DNA methylation patterns can link carbapenemase genes to pathogenic hosts and plasmid context directly from patient samples. This provides a route from rapid antimicrobial resistance gene detection to actionable public health surveillance. Data summaryAll sequencing data after human content filtering have been deposited at the European Nucleotide Archive (ENA, BioProject accession PRJEB108076, with all isolate sequencing data for mock community generation available under the sample accession numbers SAMEA121375149-58, all isolate sequencing data from the rectal swabs available at SAMEA121334008-24, all metagenomic data from the rectal swabs available at SAMEA121325220-27, and all quasimetagenomic data available at SAMEA122914816-23, SAMEA122920068-74). All code is available at GitHub: https://github.com/harikaurel/cupid. All other supporting data are provided in the article and supplementary tables.

genomics↗

bioRxiv2025

Resolving plasmid-encoded carbapenem resistance dynamics and reservoirs in a hospital setting through nanopore sequencing

The growing resistance of Enterobacterales to last-resort antibiotics such as carbapenems puts a significant burden on healthcare systems, also due to plasmids driving a rapid spread of carbapenem resistance. We here evaluate the use of long-read nanopore sequencing to investigate carbapenem resistance dynamics and the role of plasmid transfers and environmental reservoirs in the hospital setting. Over 13 months, routine clinical diagnostics identified recurring isolates of carbapenem-resistant Citrobacter species carrying Klebsiella pneumoniae carbapenemases (KPC) and/or OXA-48-like carbapenemases from patient screening and hospital drain samples. While routine diagnostic approaches provided limited insights into the carbapenem resistance dynamics, we show that near-complete de novo assembly of chromosomes and plasmids by long-read nanopore sequencing allowed for high-resolution strain identification, plasmid profiling, and antibiotic resistance gene detection. Notably, genomically nearly indistinguishable Citrobacter freundii of the high-risk sequence type ST91 genomes were recovered from screening samples collected in the same hospital room one year apart. We further provide evidence of a KPC-2 encoding IncN plasmid that is likely to have spread across bacterial species and between patient and drain isolates, which emphasizes the role of contaminated drains in the persistence and dissemination of resistances within the hospital environment. Overall, this study demonstrates the value of long-read nanopore sequencing for uncovering the complex dynamics of carbapenem resistance spread and persistence in the hospital setting, and its potential implications for Infection Prevention and Control. Impact statementThis study demonstrates how long-read nanopore sequencing can resolve the complex dynamics of plasmid-mediated antimicrobial resistance in clinical and environmental samples within the hospital setting. By linking patient- and drain-derived isolates through near-complete de novo assemblies, we reveal hidden reservoirs and dynamics behind the persistence of cabapenem resistance over extended time periods. This work shows how long-read sequencing approaches can uncover resistance dynamics that are missed using standard diagnostic methods, with implications for infection control and surveillance. Data SummaryThe study sequences are available at the National Center for Biotechnology Information (NCBI) under BioProject accession number PRJNA1297122. The raw sequence read data is available at NCBI sequence read archive (SRA (https://www.ncbi.nlm.nih.gov/sra)) under accession numbers SRR34727947-59. The chromosomal assemblies of all ST91 strains are available at NCBI GenBank under the Biosample accession numbers SAMN50449475-80. All other supporting data are provided in the article and supplementary data files.

microbiology↗