A genome-wide CRISPR/Cas9 screen identifies a role for Rab5A and early endosomes in hepatitis E virus replication.

Hepatitis E virus (HEV) is a major cause of acute hepatitis worldwide. As the other positive-strand RNA viruses, it is believed to replicate its genome in a membrane-associated replication complex. However, current understanding of the host factors required for productive HEV infection is limited and the site as well as the composition of the HEV replication complex are still poorly characterized. To identify host factors required for HEV RNA replication, we performed a genome-wide CRISPR/Cas9 screen in permissive human cell lines harboring subgenomic HEV replicons allowing for positive and negative selection. Among the validated candidates, Ras-related early endosomal protein Rab5A was selected for further characterization. siRNA-mediated silencing of Rab5A and its effectors APPL1 and EEA1, but not of the late and recycling endosome components Rab7A and Rab11A, respectively, significantly reduced HEV RNA replication. Furthermore, pharmacological inhibition of Rab5A and of dynamin-2, required for the formation of early endosomes, resulted in a dose-dependent decrease of HEV RNA replication. Colocalization studies revealed close proximity of Rab5A, the HEV ORF1 protein, corresponding to the viral replicase, as well as HEV positive- and negative-strand RNA. In conclusion, we successfully exploited CRISPR/Cas9 and selectable subgenomic replicons to identify host factors of a noncytolytic virus. This approach revealed a role for Rab5A and early endosomes in HEV RNA replication, likely by serving as a scaffold for the establishment of functional replication complexes. Our findings yield insights into the HEV life cycle and the virus-host interactions required for productive infection.

Into the Jungle of Biological Agents of Foodborne Diseases: Time to Put Some Order for the French Risk Manager.

Consumers can be exposed to many foodborne biological hazards that cause diseases with varying outcomes and incidence and, therefore, represent different levels of public health burden. To help the French risk managers to rank these hazards and to prioritize food safety actions, we have developed a three-step approach. The first step was to develop a list of foodborne hazards of health concern in mainland France. From an initial list of 335 human pathogenic biological agents, the final list of "retained hazards" consists of 24 hazards, including 12 bacteria (including bacterial toxins and metabolites), 3 viruses and 9 parasites. The second step was to collect data to estimate the disease burden (incidence, Disability Adjusted Life Years) associated with these hazards through food during two time periods: 2008-2013 and 2014-2019. The ranks of the different hazards changed slightly according to the considered period. The third step was the ranking of hazards according to a multicriteria decision support model using the ELECTRE III method. Three ranking criteria were used, where two reflect the severity of the effects (Years of life lost and Years lost due to disability) and one reflects the likelihood (incidence) of the disease. The multicriteria decision analysis approach takes into account the preferences of the risk managers through different sets of weights and the uncertainties associated with the data. The method and the data collected allowed to estimate the health burden of foodborne biological hazards in mainland France and to define a prioritization list for the health authorities.

Hepatitis E virus RNA-dependent RNA polymerase is involved in RNA replication and infectious particle production.

BACKGROUND AND AIMS: Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis worldwide. Its positive-strand RNA genome encodes three open reading frames (ORF). ORF1 is translated into a large protein composed of multiple domains and is known as the viral replicase. The RNA-dependent RNA polymerase (RDRP) domain is responsible for the synthesis of viral RNA. APPROACH AND RESULTS: Here, we identified a highly conserved α-helix located in the RDRP thumb subdomain. Nuclear magnetic resonance demonstrated an amphipathic α-helix extending from amino acids 1628 to 1644 of the ORF1 protein. Functional analyses revealed a dual role of this helix in HEV RNA replication and virus production, including assembly and release. Mutations on the hydrophobic side of the amphipathic α-helix impaired RNA replication and resulted in the selection of a second-site compensatory change in the RDRP palm subdomain. Other mutations enhanced RNA replication but impaired virus assembly and/or release. CONCLUSIONS: Structure-function analyses identified a conserved amphipathic α-helix in the thumb subdomain of the HEV RDRP with a dual role in viral RNA replication and infectious particle production. This study provides structural insights into a key segment of the ORF1 protein and describes the successful use of reverse genetics in HEV, revealing functional interactions between the RDRP thumb and palm subdomains. On a broader scale, it demonstrates that the HEV replicase, similar to those of other positive-strand RNA viruses, is also involved in virus production.

A recurrent homozygous LMNA missense variant p.Thr528Met causes atypical progeroid syndrome characterized by mandibuloacral dysostosis, severe muscular dystrophy, and skeletal deformities.

Atypical progeroid syndromes (APS) are premature aging syndromes caused by pathogenic LMNA missense variants, associated with unaltered expression levels of lamins A and C, without accumulation of wild-type or deleted prelamin A isoforms, as observed in Hutchinson-Gilford progeria syndrome (HGPS) or HGPS-like syndromes. A specific LMNA missense variant, (p.Thr528Met), was previously identified in a compound heterozygous state in patients affected by APS and severe familial partial lipodystrophy, whereas heterozygosity was recently identified in patients affected by Type 2 familial partial lipodystrophy. Here, we report four unrelated boys harboring homozygosity for the p.Thr528Met, variant who presented with strikingly homogeneous APS clinical features, including osteolysis of mandibles, distal clavicles and phalanges, congenital muscular dystrophy with elevated creatine kinase levels, and major skeletal deformities. Immunofluorescence analyses of patient-derived primary fibroblasts showed a high percentage of dysmorphic nuclei with nuclear blebs and typical honeycomb patterns devoid of lamin B1. Interestingly, in some protrusions emerin or LAP2α formed aberrant aggregates, suggesting pathophysiology-associated clues. These four cases further confirm that a specific LMNA variant can lead to the development of strikingly homogeneous clinical phenotypes, in these particular cases a premature aging phenotype with major musculoskeletal involvement linked to the homozygous p.Thr528Met variant.

Indoxyl sulfate inhibits muscle cell differentiation via Myf6/MRF4 and MYH2 downregulation.

BACKGROUND: Chronic kidney disease (CKD) is associated with a significant decrease in muscle strength and mass, possibly related to muscle cell damage by uremic toxins. Here, we studied in vitro and in vivo the effect of indoxyl sulfate (IS), an indolic uremic toxin, on myoblast proliferation, differentiation and expression of myogenic regulatory factors (MRF)-myoblast determination protein 1 (MyoD1), myogenin (Myog), Myogenic Factor 5 (Myf5) and myogenic regulatory factor 4 (Myf6/MRF4)-and expression of myosin heavy chain, Myh2. METHODS: C2C12 myoblasts were cultured in vitro and differentiated in myotubes for 7 days in the presence of IS at a uremic concentration of 200 µM. Myocytes morphology and differentiation was analyzed after hematoxylin-eosin staining. MRF genes' expression was studied using reverse transcription polymerase chain reaction in myocytes and 5/6th nephrectomized mice muscle. Myf6/MRF4 protein expression was studied using enzyme-linked immunosorbent assay; MYH2 protein expression was studied using western blotting. The role of Aryl Hydrocarbon Receptor (AHR)-the cell receptor of IS-was studied by adding an AHR inhibitor into the cell culture milieu. RESULTS: In the presence of IS, the myotubes obtained were narrower and had fewer nuclei than control myotubes. The presence of IS during differentiation did not modify the gene expression of the MRFs Myf5, MyoD1 and Myog, but induced a decrease in expression of Myf6/MRF4 and MYH2 at the mRNA and the protein level. AHR inhibition by CH223191 did not reverse the decrease in Myf6/MRF4 mRNA expression induced by IS, which rules out the implication of the ARH genomic pathway. In 5/6th nephrectomized mice, the Myf6/MRF4 gene was down-regulated in striated muscles. CONCLUSION: In conclusion, IS inhibits Myf6/MRF4 and MYH2 expression during differentiation of muscle cells, which could lead to a defect in myotube structure. Through these new mechanisms, IS could participate in muscle atrophy observed in CKD.

Countrywide multi-serotype outbreak of Salmonella Bovismorbificans ST142 and monophasic Salmonella Typhimurium ST34 associated with dried pork sausages in France, September 2020* to January 2021.

The French National Reference Centre for Escherichia coli, Shigella and Salmonella (FNRC-ESS) detected two human clusters of 33 cases (median age: 10 years; 17 females) infected by Salmonella enterica serotype Bovismorbificans, ST142, HC5_243255 (EnteroBase HierCC­cgMLST scheme) in September-November 2020 and of 11 cases (median age: 11 years; seven males) infected by S. enterica serotype 4,12:i:-, ST34, HC5_198125 in October-December 2020. Epidemiological investigations conducted by Santé publique France linked these outbreaks to the consumption of dried pork sausages from the same manufacturer. S. Bovismorbificans and S. 4,12:i:- were isolated by the National Reference Laboratory from different food samples, but both strains were identified in a single food sample only by qPCR. Three recalls and withdrawals of dried pork products were issued by the French general directorate of food of the French ministry for agriculture and food in November 2020, affecting eight supermarket chains. A notification on the European Rapid Alert System for Food and Feed and a European urgent enquiry on the Epidemic Intelligence Information System for Food and Waterborne Diseases and Zoonoses (EPIS-FWD) were launched. No cases were reported outside France. Outbreaks caused by multiple serotypes of Salmonella may go undetected by protocols in standard procedures in microbiology laboratories.

Foodborne botulism outbreak involving different nationalities during the Rugby World Cup: critical role of credit card data and rapid international cooperation, France, September 2023.

In September 2023, a severe outbreak of type B botulism with fifteen cases was linked to consumption of canned sardines at a restaurant in Bordeaux, France, during the Rugby World Cup. The cases were from seven countries. One death was recorded. Outbreak investigation using credit card data, rapid communication between health authorities of the affected countries and broad media communication allowed identification of cases and exposed persons and prevented further severe outcomes.

A large travel-associated outbreak of iatrogenic botulism in four European countries following intragastric botulinum neurotoxin injections for weight reduction, Türkiye, February to March 2023.

In March 2023, 34 associated cases of iatrogenic botulism were detected in Germany (30 cases), Switzerland (two cases), Austria (one case), and France (one case). An alert was rapidly disseminated via European Union networks and communication platforms (Food- and Waterborne Diseases and Zoonoses Network, EpiPulse, Early Warning and Response System) and the International Health Regulation mechanism; the outbreak was investigated in a European collaboration. We traced sources of the botulism outbreak to treatment of weight loss in Türkiye, involving intragastric injections of botulinum neurotoxin. Cases were traced using a list of patients who had received this treatment. Laboratory investigations of the first 12 German cases confirmed nine cases. The application of innovative and highly sensitive endopeptidase assays was necessary to detect minute traces of botulinum neurotoxin in patient sera. The botulism notification requirement for physicians was essential to detect this outbreak in Germany. The surveillance case definition of botulism should be revisited and inclusion of cases of iatrogenic botulism should be considered as these cases might lack standard laboratory confirmation yet warrant public health action. Any potential risks associated with the use of botulinum neurotoxins in medical procedures need to be carefully balanced with the expected benefits of the procedure.

Salmonella enterica subsp. enterica Welikade: guideline for phylogenetic analysis of serovars rarely involved in foodborne outbreaks.

BACKGROUND: Salmonella spp. is a major foodborne pathogen with a wide variety of serovars associated with human cases and food sources. Nevertheless, in Europe a panel of ten serovars is responsible for up to 80% of confirmed human cases. Clustering studies by single nucleotide polymorphism (SNP) core-genome phylogenetic analysis of outbreaks due to these major serovars are simplified by the availability of many complete genomes in the free access databases. This is not the case for outbreaks due to less common serovars, such as Welikade, for which no reference genomes are available. In this study, we propose a method to solve this problem. We propose to perform a core genome MLST (cgMLST) analysis based on hierarchical clustering using the free-access EnteroBase to select the most suitable genome to use as a reference for SNP phylogenetic analysis. In this study, we applied this protocol to a retrospective analysis of a Salmonella enterica serovar Welikade (S. Welikade) foodborne outbreak that occurred in France in 2016. Finally, we compared the cgMLST and SNP analyses. SNP phylogenetic reconstruction was carried out considering the effect of recombination events identified by the ClonalFrameML tool. The accessory genome was also explored by phage content and virulome analyses. RESULTS: Our findings revealed high clustering concordance using cgMLST and SNP analyses. Nevertheless, SNP analysis allowed for better assessment of the genetic distance among strains. The results revealed epidemic clones of S. Welikade circulating within the poultry and dairy sectors in France, responsible for sporadic and non-sporadic human cases between 2012 and 2019. CONCLUSIONS: This study increases knowledge on this poorly described serovar and enriches public genome databases with 42 genomes from human and non-human S. Welikade strains, including the isolate collected in 1956 in Sri Lanka, which gave the name to this serovar. This is the first genomic analysis of an outbreak due to S. Welikade described to date.

Altered action potential waveform and shorter axonal initial segment in hiPSC-derived motor neurons with mutations in VRK1.

We recently described new pathogenic variants in VRK1, in patients affected with distal Hereditary Motor Neuropathy associated with upper motor neurons signs. Specifically, we provided evidences that hiPSC-derived Motor Neurons (hiPSC-MN) from these patients display Cajal Bodies (CBs) disassembly and defects in neurite outgrowth and branching. We here focused on the Axonal Initial Segment (AIS) and the related firing properties of hiPSC-MNs from these patients. We found that the patient's Action Potential (AP) was smaller in amplitude, larger in duration, and displayed a more depolarized threshold while the firing patterns were not altered. These alterations were accompanied by a decrease in the AIS length measured in patients' hiPSC-MNs. These data indicate that mutations in VRK1 impact the AP waveform and the AIS organization in MNs and may ultimately lead to the related motor neuron disease.

Mechanisms of myostatin and activin A accumulation in chronic kidney disease.

BACKGROUND: Myostatin and activin A induce muscle wasting by activating the ubiquitin proteasome system and inhibiting the Akt/mammalian target of rapamycin pathway. In chronic kidney disease (CKD), myostatin and activin A plasma concentrations are increased, but it is unclear if there is increased production or decreased renal clearance. METHODS: We measured myostatin and activin A concentrations in 232 CKD patients and studied their correlation with estimated glomerular filtration rate (eGFR). We analyzed the myostatin gene (MSTN) expression in muscle biopsies of hemodialysis (HD) patients. We then measured circulating myostatin and activin A in plasma and the Mstn and Inhba expression in muscles, kidney, liver and heart of two CKD mice models (adenine and 5/6 nephrectomy models). Finally, we analyzed whether the uremic toxin indoxyl sulfate (IS) increased Mstn expression in mice and cultured muscle cells. RESULTS: In patients, myostatin and activin A were inversely correlated with eGFR. MSTN expression was lower in HD patients' muscles (vastus lateralis) than in controls. In mice with CKD, myostatin and activin A blood concentrations were increased. Mstn was not upregulated in CKD mice tissues. Inha was upregulated in kidney and heart. Exposure to IS did not induce Mstn upregulation in mouse muscles and in cultured myoblasts and myocytes. CONCLUSION: During CKD, myostatin and activin A blood concentrations are increased. Myostatin is not overproduced, suggesting only an impaired renal clearance, but activin A is overproduced in the kidney and heart. We propose to add myostatin and activin A to the list of uremic toxins.

Investigation of an international outbreak of multidrug-resistant monophasic Salmonella Typhimurium associated with chocolate products, EU/EEA and United Kingdom, February to April 2022.

An extensive multi-country outbreak of multidrug-resistant monophasic Salmonella Typhimurium infection in 10 countries with 150 reported cases, predominantly affecting young children, has been linked to chocolate products produced by a large multinational company. Extensive withdrawals and recalls of multiple product lines have been undertaken. With Easter approaching, widespread product distribution and the vulnerability of the affected population, early and effective real-time sharing of microbiological and epidemiological information has been of critical importance in effectively managing this serious food-borne incident.

A novel bi-allelic loss-of-function mutation in STIM1 expands the phenotype of STIM1-related diseases.

STIM1, the stromal interaction molecule 1, is the key protein for maintaining calcium concentration in the endoplasmic reticulum by triggering the Store Operated Calcium Entry (SOCE). Bi-allelic mutations in STIM1 gene are responsible for a loss-of-function in patients affected with a CRAC channelopathy syndrome in which severe combined immunodeficiency syndrome (SCID-like), autoimmunity, ectodermal dysplasia and muscle hypotonia are combined. Here, we studied two siblings from a consanguineous Syrian family, presenting with muscle weakness, hyperlaxity, elastic skin, tooth abnormalities, dysmorphic facies, hypoplastic patellae and history of respiratory infections. Using exome sequencing, we have identified a new homozygous frameshift mutation in STIM1: c.685delT [p.(Phe229Leufs*12)], leading to a complete loss of STIM1 protein. In this study, we describe an unusual phenotype linked to STIM1 mutations, combining clinical signs usually observed in different STIM1-related diseases. In particular, we confirmed that the complete loss of STIM1 function is not always associated with severe immune disorders. Altogether, our results broaden the spectrum of phenotypes associated with mutations in STIM1 and opens new perspectives on the pathological mechanisms associated with a defect in the proteins constituting the SOCE complex.

First characterization of congenital myasthenic syndrome type 5 in North Africa.

BACKGROUND: Congenital myasthenic syndromes (CMS) are associated with defects in the structure and the function of neuromuscular junctions. These rare disorders can result from mutations in the collagenic tail of endplate acetylcholinesterase (COLQ) essentially associated with autosomal recessive inheritance. With the lowered cost of genetic testing and increased access to next-generation sequencing, many mutations have been reported to date. METHODS AND RESULTS: In this study we identified the first COLQ homozygous mutation c.1193T>A in the North African population. This study outlines the genetic and phenotypic features of a CMS patient in a Moroccan family. It also describes a novel COLQ missense mutation associated with CMS-5. CONCLUSION: COLQ mutations are probably underdiagnosed in these North African populations, this is an issue as CMS-5 may be treated with ephedrine, and albuterol. Indeed, patients can seriously benefit and even recover after the treatment that should be planned according to genetic tests and clinical findings.

Splicing impact of deep exonic missense variants in CAPN3 explored systematically by minigene functional assay.

Improving the accuracy of variant interpretation during diagnostic sequencing is a major goal for genomic medicine. To explore an often-overlooked splicing effect of missense variants, we developed the functional assay ("minigene") for the majority of exons of CAPN3, the gene responsible for limb girdle muscular dystrophy. By systematically screening 21 missense variants distributed along the gene, we found that eight clinically relevant missense variants located at a certain distance from the exon-intron borders (deep exonic missense variants) disrupted normal splicing of CAPN3 exons. Several recent machine learning-based computational tools failed to predict splicing impact for the majority of these deep exonic missense variants, highlighting the importance of including variants of this type in the training sets during the future algorithm development. Overall, 24 variants in CAPN3 gene were explored, leading to the change in the American College of Medical Genetics and Genomics classification of seven of them when results of the "minigene" functional assay were considered. Our findings reveal previously unknown splicing impact of several clinically important variants in CAPN3 and draw attention to the existence of deep exonic variants with a disruptive effect on gene splicing that could be overlooked by the current approaches in clinical genetics.

Recombinant Hepatitis E Viruses Harboring Tags in the ORF1 Protein.

Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis and jaundice in the world. Current understanding of the molecular virology and pathogenesis of hepatitis E is incomplete, due particularly to the limited availability of functional tools. Here, we report the development of tagged HEV genomes as a novel tool to investigate the viral life cycle. A selectable subgenomic HEV replicon was subjected to random 15-nucleotide sequence insertion using transposon-based technology. Viable insertions in the open reading frame 1 (ORF1) protein were selected in a hepatoblastoma cell line. Functional insertion sites were identified downstream of the methyltransferase domain, in the hypervariable region (HVR), and between the helicase and RNA-dependent RNA polymerase domains. HEV genomes harboring a hemagglutinin (HA) epitope tag or a small luciferase (NanoLuc) in the HVR were found to be fully functional and to allow the production of infectious virus. NanoLuc allowed quantitative monitoring of HEV infection and replication by luciferase assay. The use of HA-tagged replicons and full-length genomes allowed localization of putative sites of HEV RNA replication by the simultaneous detection of viral RNA by fluorescence in situ hybridization and of ORF1 protein by immunofluorescence. Candidate HEV replication complexes were found in cytoplasmic dot-like structures which partially overlapped ORF2 and ORF3 proteins as well as exosomal markers. Hence, tagged HEV genomes yield new insights into the viral life cycle and should allow further investigation of the structure and composition of the viral replication complex.IMPORTANCE Hepatitis E virus (HEV) infection is an important cause of acute hepatitis and may lead to chronic infection in immunocompromised patients. Knowledge of the viral life cycle is incomplete due to the limited availability of functional tools. In particular, low levels of expression of the ORF1 protein or limited sensitivity of currently available antibodies or both limit our understanding of the viral replicase. Here, we report the successful establishment of subgenomic HEV replicons and full-length genomes harboring an epitope tag or a functional reporter in the ORF1 protein. These novel tools should allow further characterization of the HEV replication complex and to improve our understanding of the viral life cycle.

Disentangling a complex nationwide Salmonella Dublin outbreak associated with raw-milk cheese consumption, France, 2015 to 2016.

On 18 January 2016, the French National Reference Centre for Salmonella reported to Santé publique France an excess of Salmonella enterica serotype Dublin (S. Dublin) infections. We investigated to identify the source of infection and implement control measures. Whole genome sequencing (WGS) and multilocus variable-number tandem repeat analysis (MLVA) were performed to identify microbiological clusters and links among cases, animal and food sources. Clusters were defined as isolates with less than 15 single nucleotide polymorphisms determined by WGS and/or with identical MLVA pattern. We compared different clusters of cases with other cases (case-case study) and controls recruited from a web-based cohort (case-control study) in terms of food consumption. We interviewed 63/83 (76%) cases; 2,914 controls completed a questionnaire. Both studies' findings indicated that successive S. Dublin outbreaks from different sources had occurred between November 2015 and March 2016. In the case-control study, cases of distinct WGS clusters were more likely to have consumed Morbier (adjusted odds ratio (aOR): 14; 95% confidence interval (CI): 4.8-42) or Vacherin Mont d'Or (aOR: 27; 95% CI: 6.8-105), two bovine raw-milk cheeses. Based on these results, the Ministry of Agriculture launched a reinforced control plan for processing plants of raw-milk cheeses in the production region, to prevent future outbreaks.

Paediatric haemolytic uraemic syndrome related to Shiga toxin-producing Escherichia coli, an overview of 10 years of surveillance in France, 2007 to 2016.

IntroductionHaemolytic uraemic syndrome (HUS) related to Shiga toxin-producing Escherichia coli (STEC) is the leading cause of acute renal failure in young children. In France, HUS surveillance in children aged < 15 years was implemented starting from 1996.AimWe present the results of this surveillance between 2007 and 2016.MethodsA voluntary nationwide network of 32 paediatric departments notifies cases. Two national reference centres perform microbiological STEC confirmation.ResultsOver the study period, the paediatric HUS incidence rate (IR) was 1.0 per 100,000 children-years, with a median of 116 cases/year. In 2011, IR peaked at 1.3 per 100,000 children-years, and decreased to 1.0 per 100,000 children-years in 2016. STEC O157 associated HUS peaked at 37 cases in 2011 and decreased to seven cases in 2016. Cases of STEC O26-associated HUS have increased since 2010 and STEC O80 associated HUS has emerged since 2012, with 28 and 18 cases respectively reported in 2016. Four STEC-HUS food-borne outbreaks were detected (three STEC O157 linked to ground beef and raw-milk cheese and one STEC O104 linked to fenugreek sprouts). In addition, two outbreaks related to person-to-person transmission occurred in distinct kindergartens (STEC O111 and O26).ConclusionsNo major changes in HUS IRs were observed over the study period of 10 years. However, changes in the STEC serogroups over time and the outbreaks detected argue for continuing epidemiological and microbiological surveillance.

Outbreak of Salmonella enterica serotype Poona in infants linked to persistent Salmonella contamination in an infant formula manufacturing facility, France, August 2018 to February 2019.

We describe a Salmonella Poona outbreak involving 31 infant cases in France. Following outbreak detection on 18 January 2019, consumption of rice-based infant formula manufactured at a facility in Spain was identified as the probable cause, leading to a recall on 24 January. Whole genome sequencing analysis linked present outbreak isolates to a 2010-11 S. Poona outbreak in Spain associated with formula manufactured in the same facility, indicating a persistent source of contamination.

Outbreak of Shiga toxin-producing Escherichia coli (STEC) O26 paediatric haemolytic uraemic syndrome (HUS) cases associated with the consumption of soft raw cow's milk cheeses, France, March to May 2019.

We report an outbreak of Shiga toxin-producing Escherichia coli (STEC) associated paediatric haemolytic uraemic syndrome linked to the consumption of raw cow's milk soft cheeses. From 25 March to 27 May 2019, 16 outbreak cases infected with STEC O26 (median age: 22 months) were identified. Interviews and trace-back investigations using loyalty cards identified the consumption of raw milk cheeses from a single producer. Trace-forward investigations revealed that these cheeses were internationally distributed.