Abstract 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 statement Culture-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 summary All 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.

On Tuesday, September 9, 2025, the Cantonal Laboratory of Basel City received reports of cases of vomiting following the consumption of a macaroni dish served at a school canteen. The outbreak involved 27 children, and the emetic symptoms started 40-90 min after lunch. Microbiological analyses of food samples obtained from the canteen kitchen revealed the presence of B. cereus at 1100 colony-forming units (cfu)/g in leftovers of a béchamel sauce that was an ingredient of the macaroni dish. Nanopore long-read sequencing methodology and the BTyper3 tool were applied to identify B. mosaicus subsp. cereus biovar Emeticus (B. Emeticus) as the most likely etiological agent of this outbreak. Strain B. Emeticus BH-1 belonged to panC group III, sequence type (ST)164, and contained emetic toxin (cereulide) genes cesA, cesB, cesC, and cesD, but lacked enterotoxins hblD and cytK1 that cause diarrhea. This is one of the few reports of B. Emeticus ST164 implicated in food intoxication. Based on the epidemiological and microbiological investigations, the macaroni dish was identified as the most likely vehicle of transmission in this outbreak, although definitive evidence was limited by the lack of retentionsamples of the full meal. Inadequate temperature control during food holding and serving in the school canteen likely led to B. cereus proliferation and toxin production and was the most probable risk factor that contributed to the outbreak. This outbreak emphasizes the importance of considering the presence of B. cereus for implementing appropriate procedures to ensure the safety of food produced within the catering sector. Our study highlights the added value of a combined approach that used epidemiological, microbiological, and advanced next-generation whole genome sequencing (WGS) methods to identify the likely outbreak source and the etiological B. cereus strain.

Reports in the literature indicate that ice cream has been associated with illnesses linked to several pathogens, including Listeria monocytogenes. Farm-produced ice cream and sorbet are commonly marketed in Switzerland, yet data on their bacteriological quality are scarce. This study assessed the prevalence of foodborne pathogens and hygiene indicator organisms in frozen dessert (ice cream and sorbet) produced and marketed by Swiss farms. Between June and September 2025, 100 samples (ice cream, n = 61; sorbet, n = 39) were collected from 48 farms across 10 cantons.Samples were analyzed qualitatively for Salmonella, Shiga toxin-producing Escherichia coli (STEC), and Listeria spp., and quantitatively for E. coli, Staphylococcus aureus, and members of the Bacillus cereus group. Neither Salmonella, STEC, nor L. monocytogenes were detected. Escherichia coli and S. aureus counts remained below 1 log CFU/g and 2 log CFU/g, respectively. Members of the B. cereus group (B. mosaicus subsp. cereus, B. mosaicus, B. mycoides, B. cereus s.s., B. toyonensis) were identified in 15% of samples, at levels ranging from 2.00 to 3.57 log CFU/g. One isolate of Bacillus mosaicus subsp. cereus harboring the cereulide synthetase gene cluster was recovered from an almond ice cream sample (2 log CFU/g). The findings indicate an overall absence of major bacterial pathogens and low levels of indicator organisms in Swiss farm ice cream and sorbet but highlight the relevance of B. cereus group members as potential hazards requiring monitoring.

Abstract 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.

Antimicrobial resistance (AMR) is a global threat to both human and animal health. Carbapenems are last-resort antimicrobials used to treat severe infections with multidrug-resistant Gram-negative nosocomial pathogens in humans. Therefore, the dissemination of carbapenemase-producing Enterobacterales (CPE) has emerged as a major concern worldwide. Although carbapenems are not routinely used in veterinary medicine, CPE, including OXA-48-like-producing Escherichia coli, are increasingly being reported in companion animals. We document the first report of E. coli-harbouring blaOXA-484 isolated from a urine sample from a dog with a history of chronic thoracolumbar myelopathy. Using a combined Oxford Nanopore (ONT) long-reads and Illumina short-reads sequencing approach, the isolate was characterized and an IncF plasmid containing blaOXA-484 was reconstructed. The isolate belonged to sequence type (ST)167, which is an emerging high-risk clone frequently reported among human clinical isolates. The blaOXA-484 gene was harboured in a composite transposon bracketed by IS26 identical to that of blaOXA-484 carried on an IncX plasmid pOXA-484-JS316 from a human clinical E. coli ST410 from Germany. The isolation of the epidemic clone ST167 harbouring blaOXA-484 from a canine infection raises the hypothesis of a transmission event between humans and companion animals.

Background: Livestock production contributes to the emergence and spread of antimicrobial resistance (AMR), with pig farming accounting for a large share of veterinary antibiotic use. Manure application to fields can release drug-resistant bacteria and AMR genes into the environment, creating potential transmission routes to humans. Mobile genetic elements such as plasmids and transposons facilitate horizontal transfer of AMR genes between bacteria, including pathogens. However, quantitative data on the manure resistome and its links to antibiotic use remain limited. Shotgun metagenomics provides broad insights into microbiota and AMR composition, with long-read sequencing offering improved resolution of the genomic context of AMR genes. Here, we applied long-read shotgun metagenomics to investigate the diversity, abundance, and mobility potential of AMR genes in 24 manure samples from 14 Swiss pig farms with documented antibiotic use. Results: Across 24 manure samples, 225 distinct AMR genes were detected, with tetracycline resistance genes being most prevalent. Manure samples from farms reporting the highest recent antibiotic use contained greater AMR gene abundance and richness. Metagenomic assemblies revealed that 77% of AMR genes with resolved flanking regions were located near transposases, recombinases, integrases, or relaxases, suggesting high transfer potential. The tigecycline resistance gene tet(X6) and related variants were identified in 21 of 24 samples, frequently embedded within mobile genetic elements. Two samples contained complete gene clusters of the vancomycin resistance determinant vanB, one of which was part of the conjugative transposon Tn1549. In one sample, a single highly abundant plasmid encoding beta-lactam and aminoglycoside resistance accounted for 42% of the total AMR gene load. Conclusions: Pig manure is a reservoir of diverse and mobile AMR genes, including those conferring resistance to critically important antibiotics. Long-read metagenomics adds valuable genomic context, supporting AMR monitoring and risk assessment within a One Health framework.

Uropathogenic Escherichia coli (UPEC) are the leading cause of urinary tract infections (UTIs) and the virulence factor PapGII is linked to development of urosepsis. Current culture-based diagnostic assays are slow and do not include risk-assessment of the pathogen’s potential invasiveness. Therefore, clinicians rely on empirical antibiotic prescription choices, which contributes to evolution of antimicrobial resistance. We aimed to develop and validate a Loop Mediated Isothermal Amplification (LAMP)-based assay for the rapid detection of papGII -positive E. coli directly from urine. We designed specific primers targeting conserved regions of the E. coli uidA and papGII genes. To validate the assay, we collected 191 consecutive urine specimen samples over a three-day period from routine diagnostics. Culture results from routine diagnostics were used as reference for E. coli detection and whole genome sequencing for papGII detection. Our assay showed a limit of detection of 10 3 CFU/ml, allowing detection at bacterial concentrations relevant for UTI. Among the clinical urine samples tested, the assay showed 87% sensitivity and 95% specificity for E. coli , and 100% sensitivity and specificity for papGII . Notably, all results were obtained within 40 minutes and without extensive sample preparation, highlighting the potential of early UPEC identification with an increased risk of invasiveness.

Uropathogenic Escherichia coli (UPEC) are the leading cause of UTIs. The UPEC-associated virulence factor PapGII is linked to development of urosepsis. Current culture-based diagnostic assays are slow and do not include risk-assessment of the pathogen’s potential invasiveness. Therefore, clinicians rely on empirical antibiotic prescription choices, which contributes to evolution of antimicrobial resistance. We aimed to develop and validate a Loop Mediated Isothermal Amplification (LAMP)-based assay for the rapid detection of papGII-positive E. coli directly from urine.We designed specific primers targeting conserved regions of the E. coli uidA and papGII genes. Serial dilutions of targets were used to evaluate the limit of detection. To validate the assay, we collected 191 consecutive urine specimen samples over a three-day period from routine diagnostics. Culture results from routine diagnostics were used as reference for E. coli detection and whole genome sequencing for papGII detection. For each target we calculated the optimal thresholds to maximize sensitivity and specificity.Our assay showed a limit of detection of 103 CFU/ml, allowing detection at bacterial concentrations relevant for UTI. Among the clinical urine samples tested, the assay showed 87% sensitivity and 95% specificity for E. coli, and 100% sensitivity and specificity for papGII, compared to the reference methods. Notably, all results were obtained within 40 minutes and without extensive sample preparation, highlighting the potential of early UPEC identification and invasiveness risk assessment.

In veterinary medicine, the obligate intracellular bacteria Chlamydia (C.) abortus, Chlamydia caviae, and Chlamydia pecorum are known to cause ovine enzootic abortion, conjunctivitis in guinea pigs, and ocular/urogenital disease in koalas, respectively. Studying the biology of these bacteria has been challenging due to a dearth of genetic tools. This study aimed to establish stable transformation systems for C. abortus, C. pecorum, and C. caviae by introducing shuttle vectors carrying green fluorescent proteins. With the aim to select the most suitable green fluorescent protein for the tracking of chlamydiae in vitro, we further compared the fluorescence intensity of GFP to that of mNeonGreen. Transformed shuttle vectors comprised the native plasmid of the chlamydial species of interest, an Escherichia coli origin of replication (ori), a beta-lactamase (bla) or spectinomycin (aadA) resistance gene, and GFP or mNeonGreen for heterologous fluorescence expression. We compared the success of a C. suis-tailored transformation protocol (Protocol A) to that of an alternative protocol for C. psittaci and C. trachomatis (Protocol B), both of which employ calcium chloride for competence induction. Stable transformants were obtained for C. pecorum and C. caviae using protocols A and B, respectively, and we found that the fluorescence intensity of heterologously expressed GFP is higher than that of mNeonGreen. In contrast, pre-incubation with trypsin-EDTA prior to the application of calcium chloride was needed to obtain transformants of C. abortus. In summary, we established protocols for stable calcium chloride-mediated transformation for C. pecorum and C. abortus and expanded upon the genetic toolbox of C. caviae.IMPORTANCEChlamydiae are a diverse group of bacteria impacting human and animal health. Many of the veterinary species, such as Chlamydia abortus, Chlamydia caviae, and Chlamydia pecorum, which cause reproductive disorders and/or conjunctivitis, are zoonotic pathogens leading to a potentially life-threatening disease in humans. Our understanding of these species has been hampered due to a lack of genetic tools. In this study, we developed calcium chloride-mediated transformation protocols for each of these species: chlamydiae are mixed with shuttle vectors containing the complete species-specific plasmid sequence, an Escherichia coli origin of replication, and an antibiotic resistance gene for selection. We could further show that certain chlamydial species become more susceptible to genetic modification if they are pre-treated with trypsin-EDTA prior to the addition of calcium chloride and the vector of interest. Overall, we demonstrate that species-specific protocol refinement is indispensable to render chlamydiae competent for genetic transformation.

BACKGROUND:The ability to differentiate between viable and dead microorganisms in metagenomic data is crucial for various microbial inferences, ranging from assessing ecosystem functions of environmental microbiomes to inferring the virulence of potential pathogens from metagenomic analysis. Established viability-resolved genomic approaches are labor-intensive as well as biased and lacking in sensitivity. RESULTS:We here introduce a new fully computational framework that leverages nanopore sequencing technology to assess microbial viability directly from freely available nanopore signal data. Our approach utilizes deep neural networks to learn features from such raw nanopore signal data that can distinguish DNA from viable and dead microorganisms in a controlled experimental setting of UV-induced Escherichia cell death. The application of explainable artificial intelligence (AI) tools then allows us to pinpoint the signal patterns in the nanopore raw data that allow the model to make viability predictions at high accuracy. Using the model predictions as well as explainable AI, we show that our framework can be leveraged in a real-world application to estimate the viability of obligate intracellular Chlamydia, where traditional culture-based methods suffer from inherently high false-negative rates. This application shows that our viability model captures predictive patterns in the nanopore signal that can be utilized to predict viability across taxonomic boundaries. We finally show the limits of our model's generalizability through antibiotic exposure of a simple mock microbial community, where a new model specific to the killing method had to be trained to obtain accurate viability predictions. CONCLUSIONS:While the potential of our computational framework's generalizability and applicability to metagenomic studies needs to be assessed in more detail, we here demonstrate for the first time the analysis of freely available nanopore signal data to infer the viability of microorganisms, with many potential applications in environmental, veterinary, and clinical settings.

Non-typhoidal Salmonella enterica (NTS) are significant foodborne pathogens responsible for many cases of enterocolitis worldwide, with the increasing threat of antimicrobial resistance (AMR) posing a growing public health concern. Salmonella Infantis has emerged as a predominant multidrug-resistant (MDR) serotype, particularly in poultry. This study investigated the Salmonella prevalence in 200 chicken meat samples from Swiss retail stores. Six (3%) samples tested positive; all were imported, and the isolates were all identified as S. Infantis. Whole genome sequencing confirmed the presence of the pESI (plasmid of emerging S. Infantis) megaplasmid, which is associated with enhanced persistence, biofilm formation, and multidrug resistance. Statistical analysis revealed a significant correlation between Salmonella prevalence and imported, unlabeled products. These findings highlight the effectiveness of Swiss control measures in the poultry production but underscore the importance of maintaining consumer awareness to mitigate Salmonella transmission and MDR risks.

Berries are globally appreciated for their health benefits, particularly due to antioxidants, and have grown in popularity over recent decades. However, several foodborne outbreaks-mainly viral, caused by norovirus and hepatitis A-have been linked to both fresh and frozen berries. In contrast, the role of frozen berries in transmitting bacterial pathogens such as Salmonella spp., Escherichia coli, and antimicrobial-resistant bacteria (AMRB) remains underexplored. To address this, we investigated both the qualitative and quantitative presence of selected pathogens. Over a three-month period (Nov 2024-Jan 2025), 100 frozen berry samples from Swiss retailers were analyzed. One-third (n = 32) were labeled organic; 96% contained imported berries. Samples included raspberries (n = 33), blueberries (n = 18), strawberries (n = 18), blackberries (n = 3), redcurrants (n = 1), and berry mixes (n = 17), with some containing blackcurrants or sour cherries (n = 10). All samples were tested qualitatively for Salmonella spp., Shiga toxin-producing E. coli (STEC), and Listeria monocytogenes, and quantitatively for E. coli and members of the Bacillus cereus group. Extended-spectrum β-lactamase-producing Enterobacterales (ESBL-E) were also screened. Salmonella, STEC, and L. monocytogenes were not detected. E. coli counts remained below detection limits. B. cereus group members were found in 12% of samples (2.0-3.41 log CFU/g), with several strains genetically matching B. cereus s.s. Thuringiensis biopesticide strains. ESBL-E were found in 2% of samples (both Egyptian strawberries), identified by WGS as Enterobacter hormaechei carrying blaCTX-M-15 and E. coli with blaSHV-12.These findings support defining microbiological criteria for frozen berries in HACCP and routine food safety testing.

Uterine disease perturbs fertility thereby causing economic losses to the dairy enterprise worldwide. About 25-40% of dairy cattle develop metritis postpartum (Sheldon et al., 2009). 16S rRNA amplicon sequencing taxonomic identification of the en­dometrial microbiome signatures differentiate cattle harbor­ing uterine disease (Becker et al., 2023), particularly at 7 and 10 days postpartum (DPP), before clinical signs of metritis are evi­dent (Tasara et al., 2023). It is hypothesized that predominating bacterial strains inhabiting the healthy bovine uterus postpar­tum elucidate antimicrobial molecules that hinder pathogens causing endometritis. This study aims to describe predictive biosynthetic gene clusters (BGCs) present in the healthy bovine endometrial microbiome that may be protective against metritis.

Notifications of Salmonella Strathcona infections increased in Europe in 2023 prompting a multi-country outbreak investigation. We aimed to describe the epidemiology of S. Strathcona infections in 17 European countries 2011–2024, investigate the genetic relatedness of S. Strathcona isolates and identify the vehicle. Cases were persons residing in the study area and with a laboratory-confirmed S. Strathcona infection 2011–2024. Confirmed cases had a S. Strathcona isolate clustering with the outbreak reference strain in core genome multilocus sequence typing (cgMLST) within 7 allelic differences (AD) and possible cases within 8–13 AD. Probable cases had an epidemiological link to a confirmed case and non-outbreak cases had an isolate > 13 AD from the outbreak reference strain. Since 2011, 662 S. Strathcona infections have been identified: 469 confirmed, 161 probable, 13 possible and 19 non-outbreak cases. Median age of the cases was 34 years (IQR: 19–58 years) and 306 (47.5%) were notified in 2023–2024. Most sequenced isolates (469/496; 94.5%) were highly genetically related (≤ 7 AD) over time and across countries, compatible with a common source. Epidemiological and traceback investigations identified small tomatoes from Sicily as the suspect food vehicle. Stringent control measures at the source are needed to stop the contamination and prevent future cases.

This study presents the phylogenetic and antimicrobial susceptibility characterization of Mycobacterium monacense, a rare nontuberculous mycobacterium (NTM), cultured from clinical extrapulmonary samples. Eight Mycobacterium monacense isolates were identified between 2019 and 2023 in the Western Cape province of South Africa. Whole-genome sequencing (WGS) was applied to assess phylogenetic relatedness, identify virulence factors, and characterize the resistome of the isolates. Antimicrobial susceptibility testing (AST) was performed using the GenoType NTM-DR line probe assay (LPA), Sensititre minimum inhibitory concentrations (MIC) plates, and the proportional method based on critical concentrations. Spatial distribution of cases was mapped using ArcGIS software. Spatiotemporal distribution patterns indicated the presence of circulating clones confined within specific geographical areas. Plasmids coding for ferredoxin and cytochrome P450 genes were identified in one cluster, which notably lacked the chromosomal mbtH gene involved in siderophore biosynthesis for iron acquisition. In contrast, isolates grouped in a second cluster harbored the mbtH chromosomal gene but lacked these plasmid-associated elements. LPA and broth microdilution showed that all Mycobacterium monacense isolates were susceptible to aminoglycosides, fluoroquinolones, and macrolides, but generally exhibited elevated MICs against β-lactam antibiotics. Phenotypic AST indicated that drugs commonly used to treat Mycobacterium tuberculosis complex (MTBC), namely bedaquiline, linezolid, and rifampicin, are effective against Mycobacterium monacense. Mycobacterium monacense in extrapulmonary cultures accentuates the need for improved diagnostics and enhanced clinical awareness of infections with rare NTM. WGS highlights the potential significance provided by plasmid-encoded genes. Current treatment regimens for MTBC exhibit therapeutic efficacy against Mycobacterium monacense isolates.

BACKGROUND:Rhodococcus equi is an intracellular bacterial pathogen that can cause infections in various hosts, including humans and animals. Host-associated virulence plasmids have been identified as key contributors to the pathogenicity of R. equi and potentially play a role in determining the host tropism of the bacteria. The investigation of additional clinical and environmental isolates is likely to provide novel insights into the population structure, infection pathways, and drug resistance of this important pathogen. We combined whole-genome sequencing and antimicrobial-susceptibility testing of 37 selected R. equi isolates from animal, human, and environmental sources, collected in Switzerland over a 21 year period. In addition, we gathered a total of 251 whole-genome sequences and 141 multi-locus sequence (MLST) typing records from public sources. Although large geographical areas are not represented due to missing genomes we used a phylogenetic approach to define diversity patterns, distribution, and host tropism of R. equi. RESULTS:Horse isolates, irrespective of the country of isolation, exhibited distinct sequence types (ST), notably ST-1 and ST-24 among others, and carried the VAPA plasmid, implying a strain-specific affinity for particular plasmid types. Several STs including ST-62 and ST-76 associated with the VAPN plasmid included both human and ruminant isolates from Switzerland, hinting at a potential common infection source. Similarly, isolates from porcine and human sources, documented in various European countries and China, exhibited common ST, including ST-18 and ST-36, and were found to harbour VAPB plasmids upon testing, suggesting potential zoonotic implications. CONCLUSIONS:Using a genomic approach we report host-specific strains that serve as carriers of virulence-associated plasmids, indicating an adaptation strategy within distinct R. equi lineages. The existence of shared plasmid profiles between farm animals and humans suggests a common infection source. Our results contribute to an improved understanding of the global genetic diversity of virulent and environmental R. equi strains, which will benefit from additional molecular epidemiological studies including strains from unrepresented geographical areas.

Sesame products such as tahini (tahin) or halva (halwa or helva), originating from Arabic cuisine, are becoming increasingly popular in Switzerland. Pathogens, such as Salmonella, can contaminate sesame products, as evidenced by various product recalls. In this study, the occurrence of Salmonella and Bacillus cereus group members was investigated in 100 sesame products (25 sesame seeds, 16 halva, 19 different sesame pastes, 7 sesame bars, 25 hummus, and 8 other products containing sesame) collected from Swiss retail stores. None of the products were positive for Salmonella, whereas B. cereus group members could be detected with bacterial counts between 1×102 and 9×102 CFU/g in 11 out of 100 (11%) products. The 11 isolates identified by matrix-assisted laser desorption ionization-time of flight were whole-genome sequenced with Illumina technology to confirm the identity of the pathogen, determine its toxin gene profile, and perform panC typing. Most of the isolates harbored genes encoding the enterotoxins Nhe, Hbl, and CytK. The isolates were assigned to diverse B. cereus group members, including one identified as B. cytotoxicus. In addition, one of the isolates matched genetically with the Thurigiensis strain used in biopesticide products. In conclusion, none of the investigated sesame products contained significant levels of Salmonella or B. cereus group members. However, as B. cereus with pathogenic potential was detected in multiple samples, proper storage is crucial to prevent its growth and ensure consumer safety, especially for products with high water activity such as hummus.

ABSTRACT Bovine mastitis poses a significant health concern for dairy cattle and a major economic burden on the dairy industry. Klebsiella spp. are important mastitis pathogens, with previous studies identifying lactose-utilization (lacacq) and iron-acquisition (fec) systems as key determinants associated with pathogenicity. To investigate how these are acquired and shared, we genomically characterized 60 mastitis-associated Klebsiella isolates by fully resolving their chromosomes and plasmids. The isolates were identified as Klebsiella pneumoniae (n = 46), Klebsiella michiganensis (n = 8), Klebsiella quasipneumoniae (n = 3), Klebsiella grimontii (n = 2), and Klebsiella variicola (n = 1). Phylogenetic analysis revealed diverse lineages, with sporadic transmission events within and across farms. Among unique Klebsiella spp. isolates, 48/55 (87.3%) and 53/55 (96.4%) harbored lacacq and fec, respectively. In K. pneumoniae, the lacacq operon was consistently found on plasmids alongside fec. Both horizontal and vertical transfer of lacacq+ fec+ plasmids were observed. Many phylogenetically diverse mastitis isolates from distant farms carried an identical conjugative 132 kb plasmid, suggesting recent acquisitions of a single circulating plasmid as a major driver of mastitis. K. pneumoniae ST107, a globally prevalent mastitis-causing lineage, carried large non-mobilizable plasmids with fec and two lacacq operons. In contrast to K. pneumoniae, mastitis-associated K. michiganensis often carried lacacq on integrative conjugative elements and inherently harbored a chromosomal fec-like gene cluster. Few isolates possessed antimicrobial resistance genes or virulence factors linked to pathogenicity in humans. Our results provide new insights into the genomic diversity of mastitis-associated Klebsiella and the role of mobile genetic elements.IMPORTANCEUnderstanding the genetic basis of Klebsiella-induced mastitis is essential for improving prevention and control strategies. Our study reveals that the key mastitis-associated traits—lactose utilization (lacacq) and iron acquisition (fec)—are commonly encoded on plasmids. The discovery of an identical conjugative plasmid in diverse Klebsiella pneumoniae lineages highlights the potential for rapid and widespread dissemination of virulence traits, independent of clonal background. However, we also show that clonal spread—combined with the vertical inheritance of a lacacq⁺ fec⁺ plasmid—contributes to the success of K. pneumoniae ST107, a globally prevalent mastitis lineage. Together, our findings highlight the central role of mobile genetic elements in the ecology of mastitis-associated Klebsiella.

OBJECTIVE:The occurrence of extended-spectrum ß-lactamase (ESBL) producing Enterobacterales in food is of concern because of the possibility of transmission of ESBL-producers and/or blaESBL genes to humans. This study aimed to investigate the presence of ESBL-producing Enterobacterales in Swiss and imported retail chicken meat. METHODS:A total of 200 samples of different types of Swiss and imported chicken meat obtained at retail level in Switzerland were screened for ESBL-producing Enterobacterales using a selective culture medium. All non-intrinsically resistant isolates were characterised by antimicrobial susceptibility testing (AST), multilocus sequence typing (MLST), and whole genome sequencing (WGS). RESULTS:Eighteen meat samples (9%) yielded a total of 19 ESBL-producing Escherichia coli (ESBL-EC). The proportion of samples containing ESBL-EC was higher in imported meat (14/55; 25%) than in Swiss meat (4/145; 3%). Co-resistance to ciprofloxacin was highly prevalent (18/19, 95%). E. coli sequence types (STs) included ST602, ST744, ST1844, and extraintestinal pathogenic (ExPEC) E. coli ST38. The blaESBL genes comprised blaCTX-M-1 (n=7), blaCTX-M-2 (n=1), blaCTX-M-8 (n=1), blaCTX-M-55 (n=1), blaSHV-12 (n=8), and blaTEM-52B (n=1). They were chromosomally (n=2) encoded or carried on plasmids belonging to IncB/O/K/Z (n=1), IncFII (n=1), IncI1-I(Alpha) (n=9), IncX1 (n=1), and IncX3 (n=5). Many of the plasmids were identical to those detected globally in ESBL-producers associated with broilers. CONCLUSIONS:The contamination with ESBL-EC in retail chicken meat in Switzerland is moderate; nevertheless, domestic and imported chicken meat is a potential vehicle for MDR ESBL-EC and for genes conferring resistance to clinically important antimicrobials including 3rd-generation cephalosporins and fluoroquinolones.

Bacillus cytotoxicus is a thermotolerant member of the Bacillus cereus group. It has been linked to rare, but at times fatal cases of diarrheal disease and might be missed at routine diagnostic screening temperatures commonly used for the B. cereus group. The pathogen is mostly found on dehydrated foods containing potato starch or insects. How it enters the food chain or whether it persists in food producing environments is largely unknown. Increased consumption of insects and convenience foods in Europe and the lack of information on the persistence of B. cytotoxicus in food environments and its virulence demand for further characterization. In this study, we aimed to obtain a better understanding of i) the food sources of B. cytotoxicus, ii) screening temperatures needed for its isolation from food matrices, iii) cytotoxicity of the organism, and iv) its ecological niche and potential epidemiological links. To this end, 112 food samples were collected, with a focus on foods exhibiting low water activity. The samples were screened for B. cytotoxicus at 42 °C and at 50 °C. Presumptive isolates were characterized by cytK-1 toxin gene PCR for differentiation of B. cytotoxicus from other B. cereus group members. Vero cell cytotoxicity assays were performed, and selected isolates were sequenced. Our results show that screening at 42 °C might be insufficient for detecting B. cytotoxicus in foods that harbor other less thermophilic Bacillus species. When screening at 50 °C, B. cytotoxicus was detected in 23% of the food samples (n = 26 isolates). The highest prevalence was detected in mashed potato products (82%) and potato flakes (67%). In contrast, a wide range of products not containing any potato ingredients did not yield B. cytotoxicus isolates. All B. cytotoxicus isolates exhibited either low or no detectable cytotoxicity. WGS analysis revealed that a highly toxic isolate is closely related to the French outbreak strain NVH 391-98. In addition, we could show that two isolates sampled 5 years apart from the same production facility only differed by seven SNPs, making it likely that B. cytotoxicus is able to persist in production facilities over a long time. Interestingly, the reoccurring strain possessed an additional plasmid and did not show cytotoxic potential when re-isolated after 5 years.