SCOPE:The 2017 European Committee on Antimicrobial Susceptibility Testing (EUCAST) subcommittee report on the role of whole genome sequencing (WGS) in antimicrobial susceptibility testing (AST) concluded that WGS antimicrobial susceptibility prediction (WGS-ASP) was not a sufficiently robust alternative to AST to guide clinical decision making at that stage and that more evidence was required [1]. Since then, the use of WGS, bioinformatic tools, machine learning (ML)/artificial intelligence (AI), databases, and prediction approaches has greatly expanded, along with an increased knowledge of resistance mechanisms and their contribution to antimicrobial susceptibility. In response, a new EUCAST ad hoc subcommittee was established in 2024 to review the literature, with the aim of assessing the current potential and limitations of WGS-ASP. METHODS:As in the previous report, the subcommittee reviewed the literature on a 'by organism' basis but expanded the list to also include enterococci, Haemophilus influenzae, and Bacteroides fragilis in addition to those already included in the first version: Enterobacterales, Pseudomonas aeruginosa, Acinetobacter baumannii, Neisseria gonorrhoeae, Staphylococcus aureus, Streptococcus pneumoniae, Clostridioides difficile, and Mycobacterium tuberculosis. Additional sections were included to cover advances in metagenomics, other omics technologies and ML/AI. The full report was compiled and reviewed by all subcommittee members before public consultation in November 2025. CONCLUSIONS AND RECOMMENDATIONS:Significant progress has been achieved in WGS-ASP, with growing evidence supporting its ability to distinguish wild-type from non-wild-type isolates and, consequently, susceptible from resistant strains, particularly for M. tuberculosis and when clinical breakpoints align with the epidemiological cut-off (ECOFF). Despite these advances, important challenges remain before WGS-ASP can be adopted as a clinical decision-making tool. Addressing these gaps will require integrated phenotypic and genotypic surveillance to strengthen the evidence base for complex resistance mechanisms and newer antimicrobial agents, alongside comparative assessments that consider both ECOFF and clinical breakpoints. The analyses will require reference method phenotypic AST and high-quality genomic data. It is critical to ensure that datasets reflect the target populations and encompass the full spectrum of antimicrobial susceptibility, while developing unified interpretation frameworks and harmonized bioinformatics tools to standardize outputs. Robust external quality assessment schemes will be essential for clinical validation, and emerging technologies such as AI and ML offer promising avenues to enhance predictive accuracy. Finally, improvements in cost and turnaround time, coupled with evaluations of setting-specific cost-effectiveness, will be key to enabling practical implementation of WGS-ASP.

Motivation Infectious diseases continue to be a leading cause of mortality and pose a significant global health threat. Thus, the development of tools for surveillance and early detection of emerging pathogens is needed.Results We introduce PathogenFinder2, a novel, alignment-free, taxonomy-agnostic model for predicting bacterial pathogenic capacity in humans using protein language models. It outperforms previous methods, particularly for novel taxa, and provides interpretable outputs by highlighting proteins most relevant to pathogenic potential. These insights aid the identification of virulence factors, vaccine targets, and infection-related metabolic pathways. Furthermore, we introduce the Bacterial Pathogenic Capacity Landscape, which reveals patterns linked to host condition, infection site, microbial antagonism, and environmental origin.Availability The model is freely available online at https://genepi.dk/pathogenfinder2, or as a standalone program (https://github.com/genomicepidemiology/PathogenFinder2).

Tomato is a staple crop and an excellent model to study host-microbiota interactions in the plant food chain. In this study, we describe a "lab-in-the-field" approach to investigate the microbiota of field-grown tomato plants. High-throughput amplicon sequencing revealed a three-microhabitat partition, phyllosphere, rhizosphere, and root interior, differentiating host-associated communities from the environmental microbiota. An individual bacterium, classified as Acinetobacter sp., emerged as a dominant member of the microbiota at the plant-soil continuum. To gain insights into the functional significance of this enrichment, we subjected rhizosphere specimens to shotgun metagenomics. Similar to the amplicon sequencing survey, a "microhabitat effect," defined by a set of rhizosphere-enriched functions, was identified. Mobilization of mineral nutrients, as well as adaptation to salinity and polymicrobial communities, including antimicrobial resistance genes (ARGs), emerged as a functional requirement sustaining metagenomic diversification. A metagenome-assembled genome representative of Acinetobacter calcoaceticus was retrieved, and metagenomic reads associated with this species identified a functional specialization for plant-growth promotion traits, such as phosphate solubilization, siderophore production, and reactive oxygen species detoxification, which were similarly represented in a tomato genotype-independent fashion. Our results revealed that the enrichment of a beneficial bacterium capable of alleviating plant abiotic stresses appears decoupled from ARGs facilitating microbiota persistence at the root-soil interface.IMPORTANCETomatoes are at center stage in global food security due to their high nutritional value, widespread cultivation, and versatility. Tomatoes provide essential vitamins and minerals, contribute to diverse diets, and support farmer livelihoods, making them a cornerstone of sustainable food systems. Beyond direct dietary benefits, the intricate relationship between tomatoes, their associated microbiota, and antimicrobial resistance gene (ARG) is increasingly recognized. Tomato plants host diverse microbial communities in association with their organs, which influence plant health and productivity. Crop management impacts the composition and function of these communities, contributing to the prevalence of ARGs in the soil and on the plants themselves. These genes can potentially transfer to human pathogens, posing a food safety and public health risk. Understanding these complex interactions is critical for developing sustainable agricultural practices capable of mitigating the impact of climatic modifications and the global threat of antimicrobial resistance.

BACKGROUND:WGS can potentially be routinely used in clinical microbiology settings, especially with the increase in sequencing accuracy and decrease in cost. Escherichia coli is the most common bacterial species analysed in those settings, thus fast and accurate diagnostics can lead to reductions in morbidity, mortality and healthcare costs. OBJECTIVES:To evaluate WGS for diagnostics and surveillance in a collection of clinical E. coli; to examine the pool of antimicrobial resistance (AMR) determinants circulating in Denmark and the most frequent STs; and to evaluate core-genome MLST (cgMLST) and SNP-based clustering approaches for detecting genetically related isolates. METHODS:We analysed the genomes of 699 E. coli isolates collected throughout all Danish Clinical Microbiology Laboratories. We used rMLST and KmerFinder for species identification, ResFinder for prediction of AMR, and PlasmidFinder for plasmid identification. We used Center for Genomic Epidemiology MLST, cgMLSTFinder and CSI Phylogeny to perform typing and clustering analysis. RESULTS:Genetic AMR determinants were detected in 56.2% of isolates. We identified 182 MLSTs, most frequently ST-69, ST-73, ST-95 and ST-131. Using a maximum 15-allele difference as the threshold for genetic relatedness, we identified 23 clusters. SNP-based phylogenetic analysis within clusters revealed from 0 to 13 SNPs, except two cases with 111 and 461 SNPs. CONCLUSIONS:WGS data are useful to characterize clinical E. coli isolates, including predicting AMR profiles and subtyping in concordance with surveillance data. We have shown that it is possible to adequately cluster isolates through a cgMLST approach, but it remains necessary to define proper interpretative criteria.

Norovirus is a leading cause of gastroenteritis globally. Understanding the emergence, evolution, and trends in norovirus genotype/strain diversity is essential to develop sustainable strategies to guide the development of interventions. Here, we used urban wastewater samples to assess and compare the diversity of noroviruses worldwide. By using a metagenomics approach, we analyzed 327 sewage samples, collected from 62 cities across 6 continents, collected over the course of two years between 2017 and 2019. Norovirus sequences were found in >80 % of the samples, yielding 7609 contigs. We identified a total of 34 capsid genotypes and 44 polymerase P-types, predominantly belonging to genogroups known to infect humans, including: GI, GII, GIV, GVIII and GIX. Changes in diversity over time were detected, along with seasonal variations in wastewater. Some rarely reported genotypes/P-types were found to be widespread, showing the potential of wastewater surveillance to complement traditional surveillance. Phylogenetic analyses revealed that in several instances, wastewater sequences were closely related to sequences reported in outbreaks of the same region. However, novel clusters were also found in locations where fewer or no sequences from outbreaks are available (e.g., African regions). The use of metagenomic sequencing for wastewater-based epidemiology has the potential to uncover the diversity of norovirus circulating in the population, being especially helpful for those regions where classical surveillance programs are not well established.

BACKGROUND:Livestock-associated MRSA (LA-MRSA) clonal complex (CC) 398 is primarily found among pigs across Europe. However, CC398 has also been sporadically identified in several Asian countries, including Thailand. This study aimed to examine the drivers contributing to the emergence of LA-MRSA CC398 in Thailand's swine industry. METHODS:Whole-genome sequencing data of 18 LA-MRSA CC398 isolates from pigs, retail pork and swine workers in Thailand were analysed. Additionally, a total of 1197 qualified genomes from various sources in 24 countries were retrieved from Pathogenwatch. Subsequently, two SNP-based phylogenetic trees were reconstructed using maximum likelihood (n = 1215) and Bayesian inference (n = 168). RESULTS:Our findings revealed that Staphylococcus aureus isolates with the CC398-SCCmec Vc (5C2&5)-t034 genotype were closely related to European isolates, including those from Denmark. In contrast, S. aureus isolates belonging to the CC398-composite SCCmec-t034 formed a phylogenetically distinct cluster. CONCLUSIONS:This study reports that the CC398-SCCmec Vc (5C2&5)-t034 genotype identified in Thai pigs may have originated from Danish swine populations, possibly introduced into Thailand through international livestock movement. The CC398-composite SCCmec-t034 genotype was also detected in domestic pigs; however, it appears to have evolved independently from the CC398-SCCmec Vc (5C2&5)-t034 genotype. Bayesian phylogenetic analysis estimates that the common ancestor of the composite genotype emerged earlier than that of the CC398-SCCmec Vc (5C2&5)-t034 genotype, indicating a separate evolutionary origin.

The effects of sample multiplexing on the detection sensitivity of antimicrobial resistance genes (ARGs) and pathogenic bacteria in metagenomic sequencing remain underexplored in newer sequencing technologies such as Oxford Nanopore Technologies (ONT), despite its critical importance for surveillance applications. Here, we evaluate how different multiplexing levels (four and eight samples per flowcell) on two ONT platforms, GridION and PromethION, influence the detection of ARGs, bacterial taxa and pathogens. While overall resistome and bacterial community profiles remained comparable across multiplexing levels, ARG detection was more comprehensive in the four-plex setting with low-abundance genes. Similarly, pathogen detection was more sensitive in the four-plex, identifying a broader range of low abundant bacterial taxa compared to the eight-plex. However, triplicate sequencing of the same microbiomes revealed that these differences were primarily due to sequencing variability rather than multiplexing itself, as similar inconsistencies were observed across replicates. Given that eight-plex sequencing is more cost-effective while still capturing the overall resistome and bacterial community composition, it may be the preferred option for general surveillance. Lower multiplexing levels may be advantageous for applications requiring enhanced sensitivity, such as detailed pathogen research. These findings highlight the trade-off between multiplexing efficiency, sequencing depth, and cost in metagenomic studies.

Mpox is an emerging zoonotic disease that caused two public health emergencies of international concern within two years. Less is known about the interplay of microbial organisms in mpox lesions which could result in superinfections that exacerbate outcomes or delay recovery. We utilized a unified metagenomic sequencing approach involving slow-speed centrifugation and differential lysis on 19 mpox lesion swabs of hospitalized patients in South Kivu province (eastern DRC) to characterize bacteria, antimicrobial resistance genes, mpox virus (MPXV), and viral co-infections. High-quality MPXV whole-genome sequences were obtained until a Ct value of 27. Furthermore, co-infections with other clinically relevant viruses, such as varicella zoster virus and herpes simplex virus-2, were detected and confirmed by real-time PCR. In addition, metagenomic sequence analysis of the bacterial content showed the presence of bacteria associated with skin and soft tissue infection in 10 of the 19 samples analyzed. These bacteria had a high abundance of resistance genes, with possible implications for antimicrobial treatment based on the predicted antimicrobial resistance. In conclusion, we report the presence of bacterial and viral pathogens in mpox lesions and detection of widespread resistance genes to the standard antibiotic treatment. The possibility of a co-infection, including antimicrobial resistance, should be considered when discussing treatment options, along with the determination of the case-fatality ratio.IMPORTANCEThe mpox virus clade Ib lineage emerged in the eastern Democratic Republic of the Congo owing to continuous human-to-human transmission in a vulnerable patient population. A major challenge of this ongoing outbreak is its occurrence in regions with severely limited healthcare infrastructure. As a result, less is known about co-infections in affected patients. Identifying and characterizing pathogens, including their antimicrobial resistance, is crucial for reducing infection-related complications and improving antimicrobial stewardship. In this study, we applied a unified metagenomics approach to detect and characterize bacterial and viral co-infections in mpox lesions of hospitalized mpox patients in the eastern DRC.

Understanding secondary attack rates is a key knowledge gap in the ongoing clade Ib mpox virus (MPXV) outbreak in the Democratic Republic of the Congo. Here, we report the first cross-sectional serological study to investigate local MPXV clade Ib transmission in South Kivu, DRC. Seropositivity was defined as a detectable titer in a cell lysate-based screening ELISA and confirmation by virus neutralization test. Sera were collected in November and December 2023 (n = 120), and in May 2024 (n = 48) from professional sex workers (PSW) and visitors of 25 bars with reports of mpox cases. We detected serological evidence for MPXV infection in 18% and 17% of these sera, respectively, indicating that PSW played an important role in MPXV clade Ib transmission in this region. Additionally, sera from 108 direct contacts of mpox cases from 34 households were collected between September 2023 and May 2024. Serological evidence for MPXV infection was found in at least one serum sample in 50% of households, including in nine households with seropositive minors, providing evidence for close-contact household transmission. Serological studies are needed to comprehend the extent and severity of the ongoing MPXV outbreak, and may be used to guide targeted vaccination strategies, particularly for high-risk groups.

Vibrio cholerae is responsible for outbreaks in Africa, but the cause of the outbreaks remains poorly understood. Here, we report the draft genomes of four Vibrio cholerae strains isolated from individuals affected by an outbreak in Kamituga, South Kivu, the Democratic Republic of the Congo, between January and May 2024.

Antimicrobial resistance (AMR) is on one of the global priority challenges. This study explored the impact of diet alteration on AMR bacteria through metagenomic analysis during the annual vegetarian festival in Thailand in October 2019. The study investigated the effects of a 10-day shift from a regular to a vegetarian diet by collecting urban sewage from Nakhon Sawan, Surat Thani, and Bangkok before, during, and after the festival. Additionally, faecal samples from individuals in the northern city were analyzed. Using shotgun metagenomic sequencing, the samples were mapped against bacterial, AMR genes, and carbohydrate-active enzymes databases. The results revealed significant changes in AMR gene abundance and increased carbohydrate metabolism genes in sewage samples from all three cities during the festival. There was also a notable shift in the composition and diversity of bacterial species, particularly in the northern city. The total abundance of AMR genes increased during the vegetarian festival across all locations. This study highlights the correlation between a population's vegetarian diet and increased AMR in Thailand. It also demonstrates that metagenomic analysis of sewage can effectively assess the impact of dietary changes on bacterial communities and AMR at a population level, providing valuable insights for public health strategies.

In the diverse landscape of African hominids, the obligate relationship between the host and its microbiome narrates signals of adaptation and co-evolution. Sequencing 546 African hominid metagenomes, including those from indigenous Hadza and wild chimpanzees, identified similar bacterial richness and diversity surpassing those of westernized populations. While hominids share core bacterial communities, they also harbor distinct, population-specific bacterial taxa tailored to specific diets, ecology and lifestyles, differentiating non-indigenous and indigenous humans and chimpanzees. Even amongst shared bacterial communities, several core bacteria have co-diversified to fulfil unique dietary degradation functions within their host populations. These co-evolutionary trends extend to non-bacterial elements, such as mitochondrial DNA, antimicrobial resistance, and parasites. Our findings indicate that microbiome-host co-adaptations have led to both taxonomic and within taxa functional displacements to meet host physiological demands. The microbiome, in turn, transcends its taxonomic interchangeable role, reflecting the lifestyle, ecology and dietary history of its host.

In the Danish pig industry, zinc oxide, used for prevention of E. coli-related post-weaning diarrhoea in weaners, was banned in 2022 due to environmental concerns. This epidemiological study aimed to estimate the effect of the discontinued use of zinc oxide on antimicrobial usage (AMU) for weaners and finishers in Danish farms. Using farm and prescription data from 2018 to 2023 from national databases, we fitted a linear mixed-effect model to a three-level nested dataset, consisting of monthly average standardized AMU (Defined Animal Daily Doses per pig-day), on a farm (n = 4020), overseen by a veterinarian (n = 146). The most significant effect of the zinc oxide ban was seen for weaners, where AMU increased by ∼5 % on average in the first 5 months post-discontinuation, followed by a ∼17 % increase after > 5 months. A long-term effect was also seen for weaners on farms that did not use zinc oxide in the year preceding the ban, where AMU increased by ∼19 % on average > 5 months after the ban was enforced, although from a lower pre-ban AMU. The between-farm variation indicated that the effect of the absence of zinc oxide on the prevalence of post-weaning diarrhoea was significantly influenced by individual farm management practices. The random variation in AMU between farms overseen by the same veterinarian was ∼10 times higher than the estimated variation between veterinarians, indicating that antimicrobial prescriptions made by veterinarians are specific to the disease status in each farm. The estimated autocorrelation in AMU between consecutive months indicates that the constant amount of antimicrobials needed to manage the production is linked to non-varying management practices in individual farms.

In September 2023, an ongoing mpox outbreak emerged in South Kivu (Democratic Republic of the Congo) that spread to other regions and countries. Here we describe the epidemiological and genomic evolution of the outbreak between September 2023 and June 2024. Samples were collected from hospitalized patients, along with data on residence and possible exposures. Employee numbers and locations were recorded for bars with sex workers. Where possible, exposures were linked to genomic sequencing data for cluster analysis. In total, 670 cases were admitted to Kamituga General Referral Hospital from 17 health areas. Among the cases, 52.4% were in females and 47.6% in males. The majority (83.4%) were linked to professional sexual interactions. Seven deaths occurred, and three healthcare workers acquired mpox. Eight out of 14 pregnant women had fetal loss. Phylogenetic analysis revealed three clade Ib clusters. Longer branches of a sequence clustering with sequences from Kenya, Uganda, Sweden and Thailand indicate more undocumented spread. Mutations were mostly APOBEC3-type mutations indicative of sustained human-to-human transmission. No clear link between sequence cluster, bar or health area was observed. These data suggest rapid spread mostly through sexual contact within densely populated areas. The spread to neighboring countries highlights the need for extended cross-border collaboration, health education strategies focusing on sex workers, contact tracing, clinical care and surveillance.

Single-cell sequencing may serve as a powerful complementary technique to shotgun metagenomics to study microbiomes. This emerging technology allows the separation of complex microbial communities into individual bacterial cells, enabling high-throughput sequencing of genetic material from thousands of singular bacterial cells in parallel. Here, we validated the use of microfluidics and semi-permeable capsules (SPCs) technology (Atrandi) to isolate individual bacterial cells from sewage and pig fecal samples. Our method involves extracting and amplifying single bacterial DNA within individual SPCs, followed by combinatorial split-and-pool single-amplified genome (SAG) barcoding and short-read sequencing. We tested two different sequencing approaches with different numbers of SPCs from the same sample for each sequencing run. Using a deep sequencing approach, we detected 1,796 and 1,220 SAGs, of which 576 and 599 were used for further analysis from one sewage and one fecal sample, respectively. In shallow sequencing data, we aimed for 10-times more cells and detected 12,731 and 17,909 SAGs, of which we used 2,456 and 1,599 for further analysis for sewage and fecal samples, respectively. Additionally, we identified the top 10 antimicrobial resistance genes (ARGs) in both sewage and feces samples and linked them to their individual host bacterial species.

Advances in Oxford Nanopore Technologies (ONT) with the introduction of the r10.4.1 flow cell have reduced the sequencing error rates to <1%. When a reference sequence is known, this allows for accurate variant calling comparable with what is known from the second-generation short-read sequencing technologies, such as Illumina. Additionally, the longer sequence reads provided by ONT enable more efficient mappings, which means the amount of multimapping reads is reduced. However, when the correct reference is not known in advance, and the target reference is highly similar to other references, the multimapping problem is still a concern. Although the ConClave algorithm has provided an accurate solution to the multimapping problem of the second-generation short-read sequencing technologies, it is less effective when resolving the multimapping problems arising from third-generation long-read sequencing technologies. To overcome this problem, we are introducing proximity scoring of alleles, which aids the ConClave algorithm to accurately assign specific alleles from databases containing loci with a high degree of redundancy. Using multilocus sequence typing as a test case, we show that this approach matches the results obtained from sequencing data of Illumina while using limited computational resources that essentially correspond to that of today's smartphones.

Listeria monocytogenes (Lm) is a serious public health foodborne pathogen cause of listeriosis, usually in elderly, pregnant and immunocompromised people, linked to consumption of contaminated food, especially ready-to-eat (RTE) products. Different protocols can be used to detect Lm, and ISO11290-1:2017 is the reference method in Europe. Through molecular techniques such as whole genome sequencing (WGS) it is possible to discriminate between Lm strains, which are unequally distributed between clinical cases, food or food related environments, probably also due to enrichment step bias towards some Lm serogroup (IIa) compared to IVb. In the present work a set of Lm strains, detected in clinical cases and food, was investigated to define Lm strains growth ability after incubation in Half Fraser broth, and Genome Wide Association Studies (GWAS) applied to correlate the growth phenotype traits to presence of relevant genes. GWAS enabled the identification of a more relevant cassette of genes associated to a holin region of bacteriophage A118 and the determination of the distribution of relevant genes, highlighted from GWAS analysis within a population of Lm IVb and IIa.

VanHAX-mediated glycopeptide resistance has been consistently high in one of the three main sewer systems in Copenhagen, Lynetten, for +20 years. To explore this for other glycopeptide resistance genes, and whether the colonization has resulted in establishment of multiple bacterial taxa, we mapped 505 shotgun metagenomic data sets from the inlet of three sewage treatment plants to 831 different glycopeptide resistance genes. Only vanHAX and vanHBX genes were differentially abundant in Lynetten. Analyses of eight contigs suggested limited variations in the flanking regions. Proximity ligation metagenomic analysis of 12 samples from Lynetten identified 441 and 5 paired reads mapping to vanHAX and vanHBX, respectively. The other end of these reads was mapped to generated metagenomic-assembled genomes and NCBI using BLAST. vanHBX could only be linked to the phylum level (Bacillota). Plasmid analysis of vanHBX Hi-C contigs showed that these were mainly located on plasmids reported found in enterococci species. Most vanHAX-linked reads could only be linked to phylum and class level, but some reads were assigned to Enterococcus faecium (7 reads), Enterococcus faecalis (4 reads), Paenibacillus apiarius (2 reads), and Paenibacillus thiaminolyticus (27 reads). Ten of the 20 Hi-C contigs-containing vanHAX were annotated as plasmid, all reported found in Enterococcus species. This study shows that while Hi-C technology is valuable for linking antimicrobial resistance genes to bacterial taxa, it suffers from challenges in reliably mapping the linked read to a genomic region with sufficient taxonomic information. Our results also suggest that over the +20 years of colonizing a sewer system, vanHAX has not become widespread across multiple taxa, remaining primarily in E. faecalis and E. faecium, with the exception of Paenibacillus.IMPORTANCELong-term colonization of microbial communities with antimicrobial-resistant bacteria is expected to result in sharing of the resistance genes between several different bacterial taxa of the communities. We investigated microbiomes from a sewer, which have been colonized with glycopeptide-resistant bacteria harboring the mobile vanHAX gene cluster for a minimum of 20 years, using metagenomics sequencing and Hi-C. We found that despite the long-term presence in the sewer, the vanHAX genes have seemingly not disseminated widely.

We report the discovery of a persistent presence of Vibrio cholerae at very low abundance in the inlet of a single wastewater treatment plant in Copenhagen, Denmark at least since 2015. Remarkably, no environmental or locally transmitted clinical case of V. cholerae has been reported in Denmark for more than 100 years. We, however, have recovered a near-complete genome out of 115 metagenomic sewage samples taken over the past 8 years, despite the extremely low relative abundance of one V. cholerae read out of 500,000 sequenced reads. Due to the very low relative abundance, routine screening of the individual samples did not reveal V. cholerae. The recovered genome lacks the gene responsible for cholerae toxin production, but although this strain may not pose an immediate public health risk, our finding illustrates the importance, challenges, and effectiveness of wastewater-based pathogen surveillance.