Marc J. A. Stevens

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 towards 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 Contig- and Unassembled-read-based Pathogen Identification and Delineation (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 16 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 the 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.

Shiga toxin-producing Escherichia coli (STEC) causes gastrointestinal diseases and has increasingly been linked to contaminated flour, as seen in global outbreaks from 2009 to 2022. The aim of this study was to determine the occurrence and strain characteristics of STEC in flour samples collected during step controls in two mills in Switzerland. In total, 1’850 samples were collected and, after enrichment, screened for stx1 and stx2 by the Assurance GDS MPX ID assay. In mill A, 19 out of 950 collected samples tested stx-positive, with the categories wheat pearlings (n=7) and food grade wheat middlings (n=4) being most predominant. In mill B, 15 out of the 900 samples were stx-positive, with wheat pearlings (n=13) most predominant. No systematic differences were observed between the two mills. Heterogeneity was noted within samples at the same sampling level: food grade wheat middlings and semi-white flour each tested positive three times, while the corresponding duplicate samples were negative in every instance. A total of 10 STEC isolates were further characterized using whole-genome sequencing. The analysis revealed heterogeneity among the isolates, with no indications of a persistent strain within the mills. The majority of the strains were classified as low-pathogenic based on their stx subtypes and the absence of eae. Nevertheless, one isolate (O182:H25, stx2a and eae positive) was identified as a highly pathogenic strain. These findings highlight the intermittent presence of diverse STEC strains in milling fractions.

Background: Listeria monocytogenes is a foodborne pathogen that poses a significant public health risk due to the high case-fatality rate of listeriosis. Whole genome sequencing (WGS) is critical for identifying outbreak clusters and enables high-resolution characterization of isolates. Methods: We collected 335 L monocytogenes isolates from human cases of listeriosis occurring in Switzerland between 2019 and 2024. WGS was used to identify disease clusters and characterize the phylogenetic relatedness, virulence profiles, and antimicrobial resistance genes of the isolates. Results: The majority (77%) of the cases involved patients aged ≥65 years and was associated with bacteremia. The 5 major clonal complexes (CCs) were CC1, CC4, CC6, CC8, and CC388. The isolates belonged to 35 infection clusters, including 3 large outbreak clusters of 21 to 37 isolates per cluster. Nine smaller clusters included isolates highly related to food isolates obtained up to 4 years earlier. Multiple singly occurring isolates were linked to international outbreaks. WGS identified variations within key virulence factors including premature stop codons in InlA, length polymorphisms of ActA, and amino acid substitutions within PrfA. Hypervirulence associated with Listeria pathogenicity island (LIPI)-3 was found predominantly among CC1, CC4, and CC6 isolates. LIPI-4 was present in CC4 and CC388, and in infrequently occurring ST32, ST213, ST217, ST220, ST382, and ST1460. Conclusions: High-resolution typing of L monocytogenes is effective for the detection of disease clusters and for linking cases to international outbreaks. The identification of rare hypervirulent lineages demonstrates the importance of investigating the phylogenetic relatedness and virulence profiles of clinical L monocytogenes.

Bacteraemia with Streptococcus (S.) gallolyticus or other streptococci in poultry is commonly diagnosed by isolation from organs such as the liver (Sekizaki et al., 2008; Dumke et al., 2015; Crispo...

The sale of raw milk via vending machines represents a well-established distribution model in many European countries, including Switzerland. As part of this study, data on the microbiological quality of raw milk sold via vending machines in Switzerland were collected. A total of 124 raw milk samples from 124 raw milk vending machines across Switzerland were analysed. In addition to standard hygiene parameters (TVC and E. coli), the scope of the investigation particularly included foodborne pathogens as well as methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum β-lactamase (ESBL)-producing Enterobacterales. Isolates were further characterised by whole-genome sequencing. Shiga toxin-producing Escherichia coli (STEC) were detected in 3.2%, Staphylococcus aureus was detected in 12.1%, Listeria monocytogenes was detected in 2.4%, Campylobacter spp. were detected in 1.6%, Yersinia enterocolitica was detected in 29.8%, and Salmonella spp. were detected in 0% of the samples. MRSA and ESBL-producing Enterobacterales were each detected in 0.8% of samples. The results highlight the potential risk of foodborne infections associated with the consumption of untreated raw milk, as well as hygiene deficiencies linked to several raw milk vending machines. Based on the generated data, the importance of the requested heat treatment of raw milk in Switzerland is clearly underscored. Furthermore, more precise and binding guidelines for self-monitoring and the management of raw milk vending machines appear necessary.

Raw meat-based diets (RMBDs) have gained popularity over the past few years. However, there is growing concern about their safety. The study objective was to evaluate the prevalence and characteristics of the pathogens Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and Campylobacter spp. in frozen commercially sold RMBDs. One hundred raw meat-based diet products (meat originating from Switzerland, Germany, Austria, Spain, Scandinavia, South Africa, and Australia) were purchased online or in local stores and identified using culture-based methods. Isolates were further characterized by phenotypic and whole genome sequencing-based methods. In total, 11% (n = 11) of the samples tested positive for Salmonella spp., 41% (n = 41) for L. monocytogenes, 58% (n = 58) for Y. enterocolitica, and 3% (n = 3) for Campylobacter spp. Other than for Salmonella, there are no legal regulations concerning contamination of RMBDs with these zoonotic pathogens. The results of our study show that it is indicated to reconsider the requirements for the microbiological quality of RMBDs and to further raise awareness regarding the risks of handling and feeding RMBDs.

The current study addresses the critical issue of Listeria monocytogenes growth in raw sausage/meat products leading to human infections, most commonly listeriosis, which is known for its high fatality rate. This research focuses on the isolation, identification, and screening of lactic acid bacteria from various meat and fish products in Switzerland. In total, 274 lactic acid bacteria strains were isolated from 30 different products and were screened for their ability to inhibit Listeria monocytogenes growth, with 51 isolates demonstrating anti-Listeria activity at 8 °C, 15 °C, 25 °C, and 37 °C. Further experiments, using a meat model and a raw sausage challenge test, demonstrated that Leuconostoc carnosum DH25 significantly inhibited Listeria monocytogenes growth during the ripening and storage of the tested meat/sausage. This inhibitory effect was found to be attributed to the bacteriocins produced by Leuconostoc carnosum DH25 rather than factors like pH or water activity. The stability of the anti-Listeria substances was examined, revealing their resistance to temperature and pH changes, making Leuconostoc carnosum DH25 a promising protective culture for raw sausages. The genome sequencing of this strain confirms its safety, with no antibiotic resistance genes or virulence factors detected, and reveals the presence of the structural genes for the production of the bacteriocin LeucocinB-Ta11a. This study underscores the potential of LAB strains and their bacteriocins as effective tools for enhancing food safety and preventing Listeria monocytogenes growth in meat products, offering valuable insights into biocontrol strategies in the food industry.

Salmonella is an important agent of gastrointestinal disease in humans. While livestock, such as cattle, poultry, and pigs, are well-recognised animal reservoirs of Salmonella, there is a lack of data on Salmonella in edible frogs, even though frog meat is a popular food worldwide. In this study, 103 live edible Chinese frogs (Hoplobatrachus rugulosus) were collected from wet markets throughout Hong Kong. After euthanasia, faeces or cloacal swabs were examined for Salmonella. Overall, Salmonella spp. were isolated from 67 (65%, CI: 0.554–0.736) of the samples. The serotypes included S. Saintpaul (33%), S. Newport (24%), S. Bareilly (7%), S. Braenderup (4%), S. Hvittingfoss (4%), S. Stanley (10%), and S. Wandsworth (16%). Many isolates were phylogenetically related. A high number of genes encoding for resistance to clinically relevant antimicrobials, and a high number of virulence determinants, were identified. Antimicrobial susceptibility testing (AST) identified multidrug resistance (MDR) in 21% of the isolates. Resistance to ampicillin, ciprofloxacin, nalidixic acid, and tetracycline was common. These results demonstrate that a high percentage of live frogs sold for human consumption in wet markets are carriers of multidrug-resistant Salmonella. Public health recommendations for handling edible frogs should be considered, to mitigate the risk of Salmonella transmission to humans.