Biology subjects

Biggel, M.

Publications and source records attributed to Biggel, M..

5 recordsLinked to original sources

bioRxiv2026

A metagenomic framework for rapid Listeria monocytogenes surveillance in food production environments

Listeria monocytogenes remains a major foodborne pathogen with high mortality and costly persistence in food-processing environments. Established diagnostics rely on selective enrichment and single-colony isolation, which could introduce strong biases by favouring fast-growing strains or those more tolerant to enrichment broth inhibitors, while suppressing slow-growing, viable-but-nonculturable, and other co-occurring strains. This can obscure true pathogen diversity and may contribute to discrepancies between strains detected in food production environments and those associated with disease. To quantify the bias introduced by established culture-based diagnostics and to assess the potential advantage of metagenomics-based pathogen detection directly from the original sample matrix, we developed and evaluated a rapid nanopore sequencing-based metagenomic framework. We designed an artificial metagenomic community of several Listeria strains, comprising L. monocytogenes lineages I-III (including hypervirulent, persistent, and low-virulence strains), other Listeria spp., and a realistic background microbiome representative of food-processing environments. We then used this mock community to spike standard surveillance sponges and compared three workflows: (i) direct nanopore metagenomic sequencing of the original sample matrix, (ii) quasi-metagenomic sequencing after 4 h, 12 h, 24 h, or 48 h of selective enrichment, and (iii) ISO-based culture followed by whole-genome sequencing of a single presumptive L. monocytogenes isolate. We found that the culture-based approach recovered only a limited subset of strains, consistently underrepresenting diversity and failing to detect multi-strain contamination. These findings were reflected by the quasi-metagenomic results, where we found relative L. monocytogenes enrichment to be strain-dependent, indicating selective enrichment bias favouring specific strains. Metagenomics captured the full spectrum of spiked Listeria strains, enabling comprehensive strain-level resolution at all inoculation levels. We only observed relative enrichment of the L. monocytogenes strains by quasi-metagenomics compared with metagenomics after 48 h of selective enrichment. While driven primarily by the additional enrichment of L. innocua, these results suggest that quasi-metagenomics improves L. monocytogenes recovery only at the cost of a substantial reduction in speed. We finally showed that the sensitivity and accuracy of metagenomics could be improved by utilising different environmental sampling materials. We did not find any significant performance improvements from nanopore sequencing-based enrichment of L. monocytogenes through adaptive sampling approaches. We conclude that integrating long-read metagenomics into routine surveillance shows great promise to improve detection and source attribution in food safety systems.

microbiology↗

bioRxiv2026

Standalone nanopore sequencing for foodborne pathogen surveillance: a large-scale evaluation and quality control framework

Whole-genome sequencing (WGS) is central to foodborne pathogen surveillance and cross-border outbreak detection. Long-read sequencing using Oxford Nanopore Technologies (ONT) promises rapid, complete, and cost-effective genome assemblies in a single workflow. However, the adoption of standalone ONT sequencing of native DNA has been slowed by concerns that DNA modifications can compromise per-base sequencing accuracy and downstream genotyping. In this study, we evaluated ONT-only sequencing performance across 294 genetically diverse isolates representing ten major foodborne pathogens. Using the SUP@v5.2 basecalling model at 50x coverage, 97.3% (286/294) of the ONT assemblies produced identical or near-identical cgMLST profiles ([≤]3 allelic differences) as Illumina-polished hybrid assemblies. Elevated error rates were observed in four Salmonella enterica serovar Kentucky and four Listeria monocytogenes isolates and were associated with the presence of specific DNA phosphorothioation or methylation systems. Re-basecalling the same dataset with the newly released HAC@v6.0 model revealed a different error profile: although 93.5% (275/294) of assemblies remained highly accurate, all 13 isolates carrying dnd (DNA phosphorothioation) or dpd (7-deazaguanine modification) systems, including isolates of S. enterica, Cronobacter sakazakii, and Vibrio parahaemolyticus, exhibited high error rates, suggesting that such atypical modifications were not adequately represented in the models training dataset. To enable rapid identification of unreliable assemblies, we developed alpaqa, a lightweight computational tool that detects systematic nanopore assembly errors without requiring supplemental short-read data or reference genomes. By identifying affected assemblies, alpaqa provides a quality safeguard for ONT-only workflows. Masking low-quality bases in assemblies flagged by alpaqa improved cgMLST accuracy, although this reduced the number of callable loci and therefore genotyping resolution. Our findings demonstrate that standalone ONT sequencing of native DNA is sufficiently accurate for routine foodborne pathogen surveillance when combined with appropriate quality control, supporting its use in harmonised genomic surveillance frameworks. Data summaryAll sequencing data generated in this study have been submitted to the NCBI Sequence Read Archive. Accession numbers for Illumina and ONT (SUP@v5.2) reads are listed in Supplementary Table S1. Raw pod5 files from error-prone isolates have been deposited in SquiDBase (SQB000021). Alpaqa is available at github.com/MBiggel/alpaqa/. An automated ONT assembly and quality control pipeline integrating alpaqa is available at github.com/MBiggel/boap/. Impact statementThis study demonstrates that standalone Oxford Nanopore sequencing of native DNA can achieve highly accurate genotyping for routine foodborne pathogen surveillance across diverse species. We show that the remaining inaccuracies are linked to specific DNA modification systems, including phosphorothioation and 7-deazaguanine modifications, which are identified here as previously unrecognised sources of systematic sequencing errors. To address this limitation, we introduce alpaqa, a reference-free method for detecting such error-prone assemblies, providing a practical quality-control framework for ONT-only workflows. Together, these results support the reliable use of nanopore sequencing in routine genomic surveillance.

genomics↗

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↗

bioRxiv2024

Development of shuttle vector-based transformation systems for Chlamydia pecorum and Chlamydia caviae

Chlamydia (C.) abortus, C. caviae and C. pecorum are obligate intracellular, zoonotic pathogens, which have all been associated with community-acquired pneumonia in humans. C. abortus is the causative agent of enzootic ovine abortion in small ruminants and can lead to miscarriage in women. C. caviae causes conjunctivitis in guinea pigs, while C. pecorum is found in livestock, resulting in economic losses and contributing to the decline of the koala population in Australia. Studying the biology of these bacteria has been challenging due to a dearth of genetic tools. This study aimed to establish transformation systems for C. abortus and C. pecorum using shuttle vectors and to expand upon already existing protocols for C. caviae. Shuttle vectors comprised the cryptic plasmid of the chlamydial species of interest, the pUC19 origin of replication (ori), a beta-lactamase (bla), and genes that mediate heterologous expression of fluorescent proteins (GFP, mNeonGreen, mScarlet). A C. suis-tailored transformation protocol and a previously established protocol for C. psittaci, C. trachomatis and C. pneumoniae were applied. While C. pecorum and C. caviae transformation experiments were successful, transformation of C. abortus remained ineffective. Shuttle vectors yielded stable transformants over several passages in the presence and absence of selective antibiotics while the fluorescence intensity of GFP was superior compared to mNeonGreen. Finally, we co-cultured GFP- and mScarlet-expressing C. pecorum strains demonstrating that both fluorophores can be detected in the same cell or even inclusion, possibly promoting homologous recombination. These findings open new avenues into our understanding of interstrain and interspecies co-infection dynamics both in vitro and in vivo.

microbiology↗

bioRxiv2024

Oxford Nanopore's 2024 sequencing technology for Listeria monocytogenes outbreak detection and source attribution: progress and clone-specific challenges

Whole genome sequencing is an essential cornerstone of pathogen surveillance and outbreak detection. Established sequencing technologies are currently challenged by Oxford Nanopore Technologies (ONT), which offers an accessible and cost-effective alternative enabling gap-free assemblies of chromosomes and plasmids. Limited accuracy has hindered its use for investigating pathogen transmission, but recent technology updates have brought significant improvements. To evaluate its readiness for outbreak detection, we selected 78 Listeria monocytogenes isolates from diverse lineages or known epidemiological clusters for sequencing with ONTs V14 Rapid Barcoding Kit and R10.4.1 flow cells. The most accurate of several tested workflows generated assemblies with a median of one error (SNP or indel) per assembly. For 66 isolates, cgMLST profiles from ONT-only assemblies were identical to those generated from Illumina data. Eight assemblies were of lower quality with more than 20 erroneous sites each, primarily caused by methylations at the GAAGAC motif (5'-GAAG6mAC-3 / 3'-GT4mCTTC-5'). This led to inaccurate clustering, failing to group isolates from a persistence-associated clone that carried the responsible restriction-modification system. Out of 50 methylation motifs detected among the 78 isolates, only the GAAGAC motif was linked to substantially increased error rates. Our study shows that most L. monocytogenes genomes assembled from ONT-only data are suitable for high-resolution genotyping, but further improvements of chemistries or basecallers are required for reliable routine use in outbreak and food safety investigations.

genomics↗