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Whole genome sequencing is an essential cornerstone of pathogen surveillance and outbreak detection. Established sequencing technologies are currently being 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 ONT's 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, the 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'-GAAG 6mA C-3'/5'-GT 4mC TTC-3'). 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., Competing Interests: The authors declare no conflict of interest.

We traced back a nationwide outbreak of human listeriosis in Switzerland to a persisting production line contamination of a factory producing baker's yeast with Listeria monocytogenes serotype 1/2a sequence type 3141. We used whole-genome sequencing to match clinical isolates to isolates from product samples.

Purpose: We aimed to characterise Yersinia enterocolitica from human clinical specimens in Switzerland using epidemiological, microbiological and whole-genome sequencing (WGS) data., Methods: Isolates (n = 149) were collected between January 2019 and December 2023. Epidemiological data was noted and strains were characterized by biochemical and serological typing, antimicrobial susceptibility testing (AST), and WGS-based analysis., Results: Most of the isolates (86%) were from stool specimens and 52% were from male patients. The patients' median age was 28 years (range < 1-94 years). Typing assigned the isolates to bioserotype 4/O:3 (44%), biotype 1A (34%), bioserotype 2/O:9 (21%), and bioserotype 3/O:3 (1%). WGS identified Y. enterocolitica (n = 147), Y. alsatica (n = 1) and Y. proxima (n = 1). Seven isolates were multidrug resistant (MDR) and harboured plasmid pAB829 carrying aph(3″)-Ib, aph(6)-Id, and tet(Y) (n = 1), pAC120 carrying aph(6)-Id and tet(A) (n = 2), or a 12.6 kb Tn2670-like transposon containing catA1, aadA12, sul1, and qacEΔ1 (n = 4). Virulence factors (VFs) included ail (n = 99), invB, (n = 145), ystA (n = 99), ystB (n = 48) and pYV-associated VFs (n = 93). MLST and cgMLST analysis showed that BT 1A strains consisted of several STs and were highly diverse, whereas BT 2/O:9 strains were all ST12 and clustered closely, and BT 4/O:3 strains mostly belonged to ST18 but were more diverse. SNP analysis revealed two highly clonal BT 4/O:3 subpopulations with wide spatio-temporal distribution., Conclusions: Y. enterocolitica BT 1A, BT 2/O:9 and BT 4/O:3 are frequently associated with human yersiniosis in Switzerland. WGS-based subtyping of Y. enterocolitica is a powerful tool to explore the genetic diversity and the pathogenic potential of human isolates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

We describe a case of Salmonella infection caused by a sucrose-fermenting Salmonella enterica Typhimurium sequence type 12 which acquired transposon CTnscr94 carrying the sucrose operon scrKYABR. Sucrose-fermenting Salmonella are particularly challenging for culture-based detection and may lead to failure to detect Salmonella in clinical samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Study Aims: Although non-toxigenic Vibrio cholerae lack the ctxAB genes encoding cholera toxin, they can cause diarrhoeal disease and outbreaks in humans. In Switzerland, V. cholerae is a notifiable pathogen and all clinical isolates are analysed at the National Reference Laboratory for Enteropathogenic Bacteria and Listeria. Up to 20 infections are reported annually. In this study, we investigated the population structure and genetic characteristics of non-toxigenic V. cholerae isolates collected over five years., Methods:  V. cholerae isolates were serotyped and non-toxigenic isolates identified using a ctxA-specific PCR. Following Illumina whole-genome sequencing, genome assemblies were screened for virulence and antibiotic resistance genes. Phylogenetic analyses were performed in the context of 965 publicly available V. cholerae genomes., Results: Out of 33 V. cholerae infections reported between January 2017 and January 2022 in Switzerland, 31 were caused by ctxA-negative isolates. These non-toxigenic isolates originated from gastrointestinal (n = 29) or extraintestinal (n = 2) sites. They were phylogenetically diverse and belonged to 29 distinct sequence types. Two isolates were allocated to the lineage L3b, a ctxAB-negative but tcpA-positive clade previously associated with regional outbreaks. The remaining 29 isolates were placed in lineage L4, which is associated with environmental strains. Genes or mutations associated with reduced susceptibility to the first-line antibiotics fluoroquinolones and tetracyclines were identified in 11 and 3 isolates, respectively. One isolate was predicted to be multidrug resistant., Conclusions:  V. cholerae infections in Switzerland are rare and predominantly caused by lowly virulent ctxAB-negative and tcpA-negative strains. As V. cholerae is not endemic in Switzerland, cases are assumed to be acquired predominantly during travel. This assumption was supported by the phylogenetic diversity of the analysed isolates.

Campylobacter is among the most frequent agents of bacterial gastroenteritis in Europe and is primarily linked to the consumption of contaminated food. The aim of this study was to assess genomic diversity and to identify antimicrobial resistance and virulence genes of 155 Campylobacter isolated from broiler carcasses (neck skin samples) in a large-scale Swiss poultry abattoir over a three-year period. Samples originated from broilers from three different types of farming systems (particularly animal-friendly stabling (PAFS), free-range farms, and organic farms). Campylobacter jejuni (n = 127) and Campylobacter coli (n = 28) were analysed using a whole genome sequencing (WGS) approach (MiniSeq; Illumina). Sequence types (STs) were determined in silico from the WGS data and isolates were assigned into complex types (CTs) using the cgMLST SeqSphere+ scheme. Antimicrobial resistance genes were identified using the Resistance Gene Identifier (RGI), and virulence genes were identified using the virulence factor database (VFDB). A high degree of genetic diversity was observed. Many sequence types (C. jejuni ST19, ST21, ST48, ST50, ST122, ST262 and C. coli ST827) occurred more than once and were distributed throughout the study period, irrespective of the year of isolation and of the broiler farming type. Antimicrobial resistance determinants included bla OXA and tet(O) genes, as well as the T86I substitution within GyrA. Virulence genes known to play a role in human Campylobacter infection were identified such as the wlaN, cstIII, neuA1, neuB1, and neuC1. Subtyping of the Campylobacter isolates identified the occurrence of a highly clonal population of C. jejuni ST21 that was isolated throughout the three-year study period from carcasses from farms with geographically different locations and different farming systems. The high rate of genetic diversity observed among broiler carcass isolates is consistent with previous studies. The identification of a persisting highly clonal C. jejuni ST21 subtype suggests that the slaughterhouse may represent an environment in which C. jejuni ST21 may survive, however, the ecological reservoir potentially maintaining this clone remains unknown., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Here we report the genome sequence of the florfenicol-resistant Enterococcus faecalis strain 90&#95;2023 isolated from a raw-meat sausage (Finocchiona) imported from Italy to Switzerland. It has a genome of 2.75 Mbp and harbors 16 antimicrobial resistance genes, including cat A8, fexA , and a truncated optrA gene on a RepA&#95;N plasmid., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: The authors declare no conflict of interest.

Chelonians are recognized as a source of human salmonellosis through direct contact or consumption of their meat. Freshwater turtles sold for food are widely available in wet markets in Asia. In this pilot study, 50 turtles belonging to three species were randomly sampled from wet markets throughout Hong Kong. The turtles were humanely euthanised and their feces or the colon were sampled for Salmonella culture. The Salmonella isolates obtained were serotyped and examined for phenotypic antimicrobial resistance and the presence of antimicrobial resistance genes. The study reports a high prevalence (42%, 95% CI: 29.4-55.8) and considerable serotype diversity of Salmonella among turtles sold in wet markets. The most common among the 11 serotypes isolated were S . Oranienburg and S . Thompson, which have been reported in turtles previously. The serotype S . Manhattan is reported in chelonians for the first time. Resistance to streptomycin and chloramphenicol was common, despite the latter being banned from aquaculture in mainland China since 2002. Resistance against fluoroquinolones and third-generation cephalosporins which represent first-line treatment options for salmonellosis was also observed. The multidrug-resistance gene cfr is identified for the first time in Salmonella . This is a worrying finding as it indicates an expansion of the cfr reservoir and potential horizontal spread to other bacteria. The results of this study emphasize the need for close surveillance of Salmonella from turtles sold as food and better regulation of turtle farming to safeguard public health and improve animal welfare., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Colon, Lugsomya, Lam, Wahl, Parkes, Cormack, Horlbog, Stevens, Stephan and Magouras.)

Poultry feed is a leading source of Salmonella infection in poultry. In Switzerland, heat-treated feed is used to reduce Salmonella incursions into flocks in conventional poultry production. By contrast, organic feed is only treated with organic acids. In 2019, the Swiss National Reference Center for Enteropathogenic Bacteria identified the rare serovar S. Jerusalem from samples of organic soya feed. Further, in July 2020, the European Union's Rapid Alert System for Food and Feed published a notification of the detection of S. Jerusalem in soya expeller from Italy. During 2020, seven S. Jerusalem isolates from seven different poultry productions distributed over six cantons in Switzerland were reported, providing further evidence of a possible outbreak. Using whole-genome sequencing (WGS), S. Jerusalem isolates from feed and from animals in Switzerland were further characterized and compared to S. Jerusalem from organic poultry farm environments in Italy. WGS results showed that feed isolates and isolates from Swiss and Italian poultry flocks belonged to the sequence type (ST)1028, grouped in a very tight cluster, and were closely related. This outbreak highlights the risk of spreading Salmonella by feed and emphasizes the need for a heat-treatment process for feed, also in organic poultry production.

Tolerance to acid is of dual importance for the food-borne pathogen Listeria monocytogenes : acids are used as a preservative, and gastric acid is one of the first defenses within the host. There are considerable differences in the acid tolerance of strains. Here we present the transcriptomic response of acid-tolerant field strains of L. monocytogenes to HCl at pH 3.0. RNAseq revealed significant differential expression of genes involved in phosphotransferase systems, oxidative phosphorylation, cell morphology, motility, and biofilm formation. Genes in the acetoin biosynthesis pathway were upregulated, suggesting that L. monocytogenes shifts to metabolizing pyruvate to acetoin under organic acid stress. We also identified the formation of cell aggregates in microcolonies as a potential relief strategy. A motif search within the first 150 bp upstream of differentially expressed genes identified a novel potential regulatory sequence that may have a function in the regulation of virulence gene expression. Our data support a model where an excess of intracellular H+ ions is counteracted by pumping H+ out of the cytosol via cytochrome C under reduced activity of the ATP synthase. The observed morphological changes suggest that acid stress may cause cells to aggregate in biofilm microcolonies to create a more favorable microenvironment. Additionally, HCl stress in the host stomach may serve as (i) a signal to downregulate highly immunogenic flagella, and (ii) as an indicator for the imminent contact with host cells which triggers early stage virulence genes.

Here, we report the whole-genome sequences of six Listeria monocytogenes strains isolated from meat and milk products in Switzerland. All of these strains carry premature stop codons or amino acid deletions in inlA .

The aim of this study was to compare the phenotype of 40 strains of L. monocytogenes under food and host relevant stress conditions. The strains were chosen to represent food and clinical isolates and to be equally distributed between the most relevant clonal complexes for clinical and food isolates (CC1 and CC6 vs CC121 and CC9), plus one group of eight strains of rare clonal complexes. Human-associated CC1 had a faster maximal growth rate than the other major complexes, and the lag time of CC1 and CC6 was significantly less affected by the addition of 4% NaCl to the medium. Food-associated CC9 strains were hypohemolytic compared to other clonal complexes, and all strains found to be resistant to increased concentrations of benzalkonium chloride belonged to CC121 and were positive for Tn6188 carrying the qacH gene. Lactic acid affected the survival of L. monocytogenes more than HCl, and there was a distinct, strain specific pattern of acid tolerant and sensitive strains. Strains from CC6 and human clinical isolates are less resilient under acid stress than those from other complexes and from food. One strain isolated from a human patient exhibited significant growth defects across all conditions.

Ready-to-eat (RTE) raw foods harbor the risk of transmitting Listeria monocytogenes from the environment to the consumer. We isolated three strains from a facility producing RTE salad. These strains were used to perform challenge tests on different RTE salad products. Here, we present the shotgun genome sequences of all three of these strains.