Whole genome sequencing (WGS) data are increasingly used for the surveillance of foodborne infections and their genetic clusters in humans, leading to the detection of more potential outbreaks that require an intervention. We present a method based on probability theory that provides operationally relevant statistics about the size of a genetic cluster, including an estimate of the real cluster size in the population, its related uncertainty and the probability that the real size exceeds a predefined threshold. The method also allows identifying large clusters, based on the number of isolates that are genetically related, and predefined operational thresholds for their size. Finally, this method allows monitoring the evolution of the real size of an outbreak over time. We use simulated scenarios and real-life examples of previously identified genetic clusters of human campylobacteriosis in Denmark to illustrate how operational questions can be answered. We provide reference tables that the reader can apply in various contexts. Our method and findings can be used i) to quantify the real size of a foodborne outbreak with a predefined certainty; ii) to support decision making in identifying the foodborne outbreaks that require an intervention and to back up communication to authorities, the industry and the public; iii) to assess the capacities of a WGS-based surveillance system of foodborne infections to identify relevant outbreaks in consideration of the number of sequenced isolates out of the total number of reported infections.

IntroductionInfections caused by Campylobacter spp. represent a severe threat to public health worldwide. National action plans have included source attribution studies as a way to quantify the contribution of specific sources and understand the dynamic of transmission of foodborne pathogens like Salmonella and Campylobacter. Such information is crucial for implementing targeted intervention. The aim of this study was to predict the sources of human campylobacteriosis cases across multiple countries using available whole-genome sequencing (WGS) data and explore the impact of data availability and sample size distribution in a multi-country source attribution model.MethodsWe constructed a machine-learning model using k-mer frequency patterns as input data to predict human campylobacteriosis cases per source. We then constructed a multi-country model based on data from all countries. Results using different sampling strategies were compared to assess the impact of unbalanced datasets on the prediction of the cases.ResultsThe results showed that the variety of sources sampled and the quantity of samples from each source impacted the performance of the model. Most cases were attributed to broilers or cattle for the individual and multi-country models. The proportion of cases that could be attributed with 70% probability to a source decreased when using the down-sampled data set (535 vs. 273 of 2627 cases). The baseline model showed a higher sensitivity compared to the down-sampled model, where samples per source were more evenly distributed. The proportion of cases attributed to non-domestic source was higher but varied depending on the sampling strategy. Both models showed that most cases could be attributed to domestic sources in each country (baseline: 248/273 cases, 91%; down-sampled: 361/535 cases, 67%;).DiscussionThe sample sizes per source and the variety of sources included in the model influence the accuracy of the model and consequently the uncertainty of the predicted estimates. The attribution estimates for sources with a high number of samples available tend to be overestimated, whereas the estimates for source with only a few samples tend to be underestimated. Reccomendations for future sampling strategies include to aim for a more balanced sample distribution to improve the overall accuracy and utility of source attribution efforts.

Campylobacter is the most common cause of bacterial foodborne illness in the EU, primarily linked to poultry. To better understand its transmission dynamics, we applied whole-genome sequencing (WGS) to Campylobacter isolates collected at slaughterhouses over a two-year period from broilers originating from 26 Danish farms. The samples included cloacal swabs and boot sock samples from broiler houses and surrounding farm environments. We identified 150 distinct cgMLST types among 883 isolates. While most cgMLST types were flock-specific, some persisted across production cycles or appeared at different farms, indicating entrenched contamination or potential common-source introductions. Notably, 39% of broiler-associated cgMLST types overlapped with human clinical isolates from the same period, with the strongest overlap among persistent and cross-farm types, particularly in conventional production systems. Our findings underscore the need for strengthened biosecurity, targeted surveillance of high-risk genotypes, and real-time WGS integration to mitigate the burden of human Campylobacteriosis. This study supports a One Health approach to managing zoonotic risk in poultry production.

Non-typhoidal Salmonella is the second most frequently reported zoonotic pathogen in the European Union and European Economic Area. Most human infections are caused by serovars Enteritidis and Typhimurium. Genomic characterisation of Salmonella isolates from humans and animals has become a routine public health surveillance tool in many countries. In this study, the relative contributions of several potential sources of human infection of the five frequently reported Salmonella serovars were estimated using machine-learning methods based on a large, cross-sectional collection of genomes from human cases, and animal and environmental sources, across ten European countries. To define the population structure, core-genome Multilocus Sequence Typing was performed. A supervised machine-learning approach was applied for source attribution in the form of a Random Forest classifier. The source and country attribution models achieved moderate accuracy (F1=0.6-0.9), which is lower than in previous studies using machine-learning on Whole Genome Sequencing data. However, attributions of human clinical isolates to different sources were generally in line with previous findings for these five serovars. While the lack of clonality in some sources hindered their prediction, it is also likely that certain sources (e.g., pets) do not serve as major contributors to human infection. Therefore, in most cases attributing these sources to the livestock species they are typically associated with, is likely appropriate. Country attributions showed that substantial human cases are attributable to countries other than their own, indicating geographical interrelatedness of sources. This highlights the value of internationally harmonised Salmonella-control policies in the food production chain.

In July-October 2025, two concurrent Campylobacter jejuni outbreaks (clones ST49#3 and ST52#4) with 112 notified cases were detected through Denmark's whole genome sequencing surveillance programme. The outbreaks were clinically severe: 45 (40%) infected individuals were hospitalised and 16 (14%) had bacteraemia. We estimated 900 laboratory-confirmed outbreak cases. Both outbreaks originated from Danish-produced chicken meat. These outbreaks reveal the vulnerabilities in the current prevention and control framework given by the regulatory tolerance for Campylobacter in fresh poultry meat.

Implementation of molecular detection methodology of Shiga toxin-producing Escherichia coli (STEC) in Danish patients began in 1997. Since then, changes in molecular detection methods and diagnostic criteria have led to the present situation, in which almost all diarrhoeal stool specimens are examined for STEC. Whole genome sequencing (WGS) of STEC isolates referred to the national reference laboratory has increased the detailed characterisation, and revealed a large spectrum, of STEC types, including cross-over pathotypes typically associated with extraintestinal disease or traveller’s diarrhoea. Association of subtype stx2a (and stx2d) with the risk of developing haemolytic uraemic syndrome (HUS) was confirmed. These changes have resulted in an increase in the number of diagnosed STEC cases from 31 cases in 1997 to 1432 in 2023. Similar increases in Europe have also been recorded. Culture of STEC is, on the other hand, declining, which poses a challenge to the identification of multiple STEC infections and outbreaks. Syndromic (PCR) test panels have also resulted in an increase in the detection of multiple microorganisms. Double or triple infections have increased the role of clinical microbiologists in interpreting and assessing the significance of diagnostic results and have also increased the need for high-quality curation of surveillance data.

Whole-genome sequencing (WGS) is increasingly used as the primary typing method for foodborne disease surveillance. It offers high-resolution cluster analysis, interoperability, and comprehensive pathogen characterization. However, implementing WGS-based foodborne surveillance also poses challenges. This paper outlines these challenges and provides practical recommendations. It requires a business plan that details the financial, technical and human resources needed, since setting up WGS-based surveillance requires substantial initial investments. During the initial phase, the per sample costs of WGS are likely higher than with traditional typing method. However, this will align or even go below that when fully transitioned to WGS-based surveillance because WGS data can be used for multiple purposes such as (sero)typing and antimicrobial and virulence characterization. It is advisable to start with a single pathogen to establish a solid foundation, with the aim of having one institutional sequencing facility. Validating accuracy and consistency of results is crucial before expanding to other pathogens. While cross-disciplinary collaboration has always played an important role in foodborne surveillance, the complexity of WGS results now makes it essential for transforming findings into effective interventions. Despite its challenges, advancements in technology and computation capabilities have made it increasingly accessible, ultimately improving public health surveillance and response.

Klebsiella pneumoniae species complex (KpSC) members, including the most important species K. pneumoniae (phylogroup Kp1 of the KpSC), are important opportunistic pathogens that display increasing rates of antimicrobial resistance worldwide. As they are widespread in food and the environment, there is a need for fast, sensitive, and reliable methods to detect KpSC members in complex matrices. Previously, the ZKIR real-time PCR assay was developed to detect all KpSC members without distinction. Given that Kp1 is the clinically most significant phylogroup of the KpSC, here, we aimed to simultaneously identify Kp1 while detecting all KpSC members. Three TaqMan probes were developed and used: the Kp1 probe to specifically identify this phylogroup; the zkir P1 probe to detect phylogroups Kp1-Kp5 and Kp7; and the zkir P2 probe to detect phylogroup Kp6. The new triplex assay was tested on a total of 95 KpSC and 19 non-KpSC strains from various sources, representing the different phylogroups as defined by whole genome sequencing. The results showed almost complete specificity, as the expected PCR results were obtained for 112 (98%) strains. The new triplex real-time PCR assay, called ZKIR-T, enables the detection of all KpSC taxa while discriminating Kp1, which will be useful for rapid screening and to focus downstream analyses on chosen phylogroups of the KpSC. IMPORTANCE:The pathogens of the Klebsiella pneumoniae species complex are widespread in food and animals and are among the main pathogens responsible for multidrug resistant infections in humans. In this study, we developed a highly sensitive detection assay that enables detection of this group of bacteria, with the simultaneous identification of the most common and clinically important species. This triplex one-reaction assay was shown to be highly sensitive and precise, enabling fast screening of varied samples for the presence of KpSC and K. pneumoniae sensu stricto.

ABSTRACT We investigated whether the introduction of polymerase chain reaction (PCR) to replace culture as the primary diagnostic method for Campylobacter species in the Danish Departments of Clinical Microbiology (DCMs) was associated with a systematic change in the number of reported episodes of human campylobacteriosis from 2015 to 2022. We described a hypothetical scenario where PCR was solely used from 2018 to 2021. We analyzed the effect of primary diagnostic methods (culture or PCR) on the number of weekly episodes of human campylobacteriosis in a negative binomial regression adjusting for time, seasonality, COVID-19 restrictions, and DCMs. Furthermore, we applied the estimated PCR effect to the number of episodes that was identified using culture. Overall, PCR was associated with a 43% estimated increase of weekly campylobacteriosis episodes [rate ratio (RR) 1.43, 95% confidence interval (95% CI) 1.34–1.53]. Up to 17%–25% additional episodes would have been reported yearly from 2018 to 2021, had PCR been solely used. Through the lens of laboratory diagnostic methods, we described a systematic change in the number of reported episodes of human campylobacteriosis in Denmark. This is operationally relevant as considerably more episodes would have been identified had PCR been solely used. Changes in diagnostic methods should be considered in the epidemiological analysis of historical data. IMPORTANCE This study is important because it shows the potential epidemiological silence associated with the use of culture as the primary diagnostic method for the laboratory identification of human campylobacteriosis. Also, we show how polymerase chain reaction methods are associated with a systematic increase in the number of human campylobacteriosis episodes as reported by routine disease surveillance. These findings are operationally relevant and have public health implications because they tell how crucial it is to consider changes in diagnostic methods, e.g., in the epidemiological analysis of historical data and in the interpretation of future data in light of the past. We also believe that this study highlights how the synergy between microbiology and epidemiology is essential for disease surveillance.

This review summarises the current knowledge of whole genome sequencing (WGS) which has become the standard method for genetic characterisation of bacteria in surveillance and outbreak investigation. Although the method offers many advantages, its use in outbreak investigations is hampered by the relatively slow turn-around time. Using new approaches to perform WGS, typing and gene detection can now be completed within one day. This break-through allows clinical consequences to be taken almost immediately after detection of relevant bacteria and may have a huge impact on the future prevention of transmission of infectious diseases.

Members of the Campylobacter lari group are causative agents of human gastroenteritis and are frequently found in shellfish, marine waters, shorebirds, and marine mammals. Within a One Health context, we used comparative genomics to characterize isolates from a diverse range of sources and geographical locations within Europe and Australia and assess possible transmission of food, animal, and environmental isolates to the human host. A total of 158 C. lari isolates from Australia, Denmark, France, and Germany, which included 82 isolates from human stool and blood, 12 from food, 14 from domestic animal, 19 from waterbirds, and 31 from the environment were analyzed. Genome-wide analysis of the genetic diversity, virulence, and antimicrobial resistance (AMR) traits was carried-out. Most of the isolates belonged to C. lari subsp. lari (Cll; 98, 62.0%), while C. lari subsp. concheus and C. lari urease-positive thermotolerant Campylobacter (UPTC) were represented by 12 (7.6%) and 15 (9.5%) isolates, respectively. Furthermore, 33 (20.9%) isolates were not assigned a subspecies and were thus attributed to distant Campylobacter spp. clades. Whole-genome sequence-derived multilocus sequence typing (MLST) and core-genome MLST (cgMLST) analyses revealed a high genetic diversity with 97 sequence types (STs), including 60 novel STs and 14 cgMLST clusters (≤10 allele differences), respectively. The most prevalent STs were ST-21, ST-70, ST-24, and ST-58 (accounting for 13.3%, 4.4%, 3.8%, and 3.2% of isolates, respectively). A high prevalence of the 125 examined virulence-related loci (from 76.8 to 98.4% per isolate) was observed, especially in Cll isolates, suggesting a probable human pathogenicity of these strains. IMPORTANCE Currently, relatedness between bacterial isolates impacting human health is easily monitored by molecular typing methods. These approaches rely on discrete loci or whole-genome sequence (WGS) analyses. Campylobacter lari is an emergent human pathogen isolated from diverse ecological niches, including fecal material from humans and animals, aquatic environments, and seafood. The presence of C. lari in such diverse sources underlines the importance of adopting an integrated One Health approach in studying C. lari population structure for conducting epidemiological risk assessment. This retrospective study presents a comparative genomics analysis of C. lari isolates retrieved from two different continents (Europe and Australia) and from different sources (human, domestic animals, waterbirds, food, and environment). It was designed to improve knowledge regarding C. lari ecology and pathogenicity, important for developing effective surveillance and disease prevention strategies.

Bacteria of the Klebsiella pneumoniae species complex (KpSC) are ubiquitous, and K. pneumoniae is a leading cause of antibiotic-resistant infections in humans. Despite the urgent public health threat represented by K. pneumoniae, there is a lack of knowledge of the contribution of food sources to colonization and subsequent infection in humans. ABSTRACT The Klebsiella pneumoniae species complex (KpSC) is a leading cause of multidrug-resistant human infections. To better understand the potential contribution of food as a vehicle of KpSC, we conducted a multicentric study to define an optimal culture method for its recovery from food matrices and to characterize food isolates phenotypically and genotypically. Chicken meat (n = 160) and salad (n = 145) samples were collected in five European countries and screened for the presence of KpSC using culture-based and zur-khe intergenic region (ZKIR) quantitative PCR (qPCR) methods. Enrichment using buffered peptone water followed by streaking on Simmons citrate agar with inositol (44°C for 48 h) was defined as the most suitable selective culture method for KpSC recovery. A high prevalence of KpSC was found in chicken meat (60% and 52% by ZKIR qPCR and the culture approach, respectively) and salad (30% and 21%, respectively) samples. Genomic analyses revealed high genetic diversity with the dominance of phylogroups Kp1 (91%) and Kp3 (6%). A total of 82% of isolates presented a natural antimicrobial susceptibility phenotype and genotype, with only four CTX-M-15-producing isolates detected. Notably, identical genotypes were found across samples—same food type and same country (15 cases), different food types and same country (1), and same food type and two countries (1)—suggesting high rates of transmission of KpSC within the food sector. Our study provides a novel isolation strategy for KpSC from food matrices and reinforces the view of food as a potential source of KpSC colonization in humans. IMPORTANCE Bacteria of the Klebsiella pneumoniae species complex (KpSC) are ubiquitous, and K. pneumoniae is a leading cause of antibiotic-resistant infections in humans. Despite the urgent public health threat represented by K. pneumoniae, there is a lack of knowledge of the contribution of food sources to colonization and subsequent infection in humans. This is partly due to the absence of standardized methods for characterizing the presence of KpSC in food matrices. Our multicentric study provides and implements a novel isolation strategy for KpSC from food matrices and shows that KpSC members are highly prevalent in salads and chicken meat, reinforcing the view of food as a potential source of KpSC colonization in humans. Despite the large genetic diversity and the low levels of resistance detected, the occurrence of identical genotypes across samples suggests high rates of transmission of KpSC within the food sector, which need to be further explored to define possible control strategies.

Background Since 2008, Danish national surveillance of Clostridioides difficile has focused on binary toxin-positive strains in order to monitor epidemic types such as PCR ribotype (RT) 027 and 078. Additional surveillance is needed to provide a more unbiased representation of all strains from the clinical reservoir. Aim Setting up a new sentinel surveillance scheme for an improved understanding of type distribution relative to time, geography and epidemiology, here presenting data from 2016 to 2019. Methods For 2─4 weeks in spring and autumn each year between 2016 and 2019, all 10 Danish Departments of Clinical Microbiology collected faecal samples containing toxigenic C. difficile. Isolates were typed at the national reference laboratory at Statens Serum Institut. The typing method in 2016–17 used tandem-repeat-sequence typing, while the typing method in 2018–19 was whole genome sequencing. Results During the study period, the sentinel surveillance scheme included ca 14–15% of all Danish cases of C. difficile infections. Binary toxin-negative strains accounted for 75% and 16 of the 20 most prevalent types. The most common sequence types (ST) were ST2/13 (RT014/020) (19.5%), ST1 (RT027) (10.8%), ST11 (RT078) (6.7%), ST8 (RT002) (6.6%) and ST6 (RT005/117) (5.1%). The data also highlighted geographical differences, mostly related to ST1 and temporal decline of ST1 (p = 0.0008) and the increase of ST103 (p = 0.002), ST17 (p = 0.004) and ST37 (p = 0.003), the latter three binary toxin-negative. Conclusion Sentinel surveillance allowed nationwide monitoring of geographical differences and temporal changes in C. difficile infections in Denmark, including emerging types, regardless of binary toxin status.

The bacterial foodborne pathogen Listeria monocytogenes clonal complex 1 (Lm-CC1) is the most prevalent clonal group associated with human listeriosis and is strongly associated with cattle and dairy products. Here, we analyze 2021 isolates collected from 40 countries, covering Lm-CC1 first isolation to present days, to define its evolutionary history and population dynamics. We show that Lm-CC1 spread worldwide from North America following the Industrial Revolution through two waves of expansion, coinciding with the transatlantic livestock trade in the second half of the 19th century and the rapid growth of cattle farming and food industrialization in the 20th century. In sharp contrast to its global spread over the past century, transmission chains are now mostly local, with limited inter- and intra-country spread. This study provides an unprecedented insight into L. monocytogenes phylogeography and population dynamics and highlights the importance of genome analyses for a better control of pathogen transmission.

Background: Campylobacter is one of the most frequent causes of bacterial gastroenteritis. Campylo bacter outbreaks are rarely reported, which could be a reflection of a surveillance without routine molecular typing. We have previously shown that numerous small outbreak-like clusters can be detected when whole genome sequencing (WGS) data of clinical Campylobacter isolates was applied. Aim: Typing based surveillance of Campylobacter infections was initiated in 2019 to enable detection of large clusters of clinical isolates and to match them to concurrent retail chicken isolates in order to react on ongoing outbreaks. Methods: We performed WGS continuously on isolates from cases (n = 701) and chicken meat (n = 164) throughout 2019. Core genome multilocus sequence typing was used to detect clusters of clinical isolates and match them to isolates from chicken meat. Results: Seventy-two clusters were detected, 58 small clusters (2-4 cases) and 14 large clusters (5-91 cases). One third of the clinical isolates matched isolates from chicken meat. One large cluster persisted throughout the whole year and represented 12% of all studied Campylobacter cases. This cluster type was detected in several chicken samples and was traced back to one slaughterhouse, where interventions were implemented to control the outbreak. Conclusion: Our WGS-based surveillance has contributed to an improved understanding of the dynamics of the occurrence of Campylobacter strains in chicken meat and the correlation to clusters of human cases.

Phages infecting Campylobacter jejuni are considered a promising intervention strategy at broiler farms, yet phage sensitivity of naturally occurring poultry isolates is not well studied. Here, we investigated phage sensitivity and identified resistance mechanisms of C. jejuni strains originating from Danish broilers belonging to the most prevalent MLST (ST) types. Determining plaque formation of 51 phages belonging to Fletchervirus or Firehammervirus showed that 21 out of 31 C. jejuni strains were susceptible to at least one phage. While C. jejuni ST-21 strains encoded the common phase variable O-methyl phosphoramidate (MeOPN) receptor of the Fletchervirus and were only infected by these phages, ST-45 strains did not encode this receptor and were exclusively infected by Firehammervirus phages. To identify internal phage resistance mechanism in ST-21 strains, we performed comparative genomics of two strains, CAMSA2002 sensitive to almost all Fletchervirus phages and CAMSA2038, resistant to all 51 phages. The strains encoded diverse clustered regularly interspaced short palindromic repeats (CRISPR) spacers but none matched the tested phages. Sequence divergence was also observed in a predicted SspE homolog and putative restriction modification systems including a methyl-specific McrBC endonuclease. Furthermore, when mcrB was deleted, CAMSA2038 became sensitive to 17 out of 43 phages, three being Firehammervirus phages that otherwise did not infect any ST-21 strains. Yet, 16 phages demonstrated significantly lower efficiencies of plating on the mcrB mutant suggesting additional resistance mechanism still restricting phage propagation in CAMSA2038. Thus, our work demonstrates that C. jejuni isolates originating from broilers may have acquired several resistance mechanisms to successfully prevent phage infection in their natural habitat.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis globally, and infections are often transmitted through consumption of raw or undercooked poultry. Campylobacter jejuni ST50 is among the top ten sequence types (STs) reported in the collected isolates listed at PubMLST records from poultry, food and clinical sources for Asia, Europe, North America, Oceania and South America. This study was designed to determine the most commonly reported C. jejuni STs globally using the PubMLST database and assess similarities between genomes of C. jejuni ST50 isolates from geographically distinct locations. To gain a better understanding of C. jejuni diversity, we compared draft genome sequences of 182 ST50 isolates recovered from retail or caecal poultry samples in Oceania, Europe and North America that were collected over a period of 9 years (2010 to 2018). Overall, phylogenetic analysis revealed that isolates from geographically distinct locations tended to cluster based on the continent where the sample was collected. Among ST50 isolates from Europe and North America, we identified resistance determinants associated with phenotypic resistance to beta-lactams (EU: 55%; GB: 43.1%), tetracyclines (CA: 77.3%; EU: 37.5%; GB: 9.8%; US: 43.5%) and fluoroquinolones (EU: 60.0%; GB: 15.7%); no resistance determinants were identified in isolates from Australia. In general, the majority of the virulence genes, with rare exceptions such as wlaN, cj1138, hddA and rfbC, were evenly distributed throughout the genomes of all ST50 isolates in this study. Genomic-based characterization of C. jejuni ST50 isolates from poultry on three continents highlighted that geographically distinct isolates have evolved independently but only represent a glimpse into the diversity of C. jejuni.

Salmonella enterica subspecies enterica serovar Typhimurium and its monophasic variant are among the most common Salmonella serovars associated with human salmonellosis each year. Related infections are often due to consumption of contaminated meat of pig, cattle and poultry origin. In order to evaluate novel microbial subtyping methods for source attribution, an approach based on weighted networks was applied on 141 human and 210 food and animal isolates of pigs, broilers, layers, ducks and cattle collected in Denmark from 2013 to 2014. A whole-genome SNP calling was performed along with cgMLST and wgMLST. Based on these genomic input data, pairwise distance matrices were built and used as input for construction of a weighted network where nodes represent genomes and links distances. Analyzing food and animal genomes, the coherence of source clustering was 90% for animal source, 85% for country, 82% for serotype and 65 % for year of isolation independently from the type of input data, suggesting animal source as the first driver of clustering formation. Adding human isolate genomes to the network, a percentage between 93.6% and 95% clustered with the existing component and only a percentage between 5% and 6.4% appeared as not attributed to any animal source. The majority of human genomes were attributed to pigs with probabilities ranging from 83.9 to 84.5%, followed by broilers, ducks, cattle and layers in descending order. In conclusion, weighted network approach based on pairwise SNPs, cgMLST and wgMLST matrices showed promising results for source attribution studies.