LiSEQ - whole-genome sequencing of a cross-sectional survey of Listeria monocytogenes in ready-to-eat foods and human clinical cases in Europe.

We present the LiSEQ (Listeria SEQuencing) project, funded by the European Food Safety Agency (EFSA) to compare Listeria monocytogenes isolates collected in the European Union from ready-to-eat foods, compartments along the food chain (e.g. food-producing animals, food-processing environments) and humans. In this article, we report the molecular characterization of a selection of this data set employing whole-genome sequencing analysis. We present an overview of the strain diversity observed in different sampled sources, and characterize the isolates based on their virulence and resistance profile. We integrate into our analysis the global L. monocytogenes genome collection described by Moura and colleagues in 2016 to assess the representativeness of the LiSEQ collection in the context of known L. monocytogenes strain diversity.

Retrospective validation of whole genome sequencing-enhanced surveillance of listeriosis in Europe, 2010 to 2015.

Background and aimThe trend in reported case counts of invasive Listeria monocytogenes (Lm), a potentially severe food-borne disease, has been increasing since 2008. In 2015, 2,224 cases were reported in the European Union/European Economic Area (EU/EEA). We aimed to validate the microbiological and epidemiological aspects of an envisaged EU/EEA-wide surveillance system enhanced by routine whole genome sequencing (WGS). Methods: WGS and core genome multilocus sequence typing (cgMLST) were performed on isolates from 2,726 cases from 27 EU/EEA countries from 2010-15. Results: Quality controls for contamination, mixed Lm cultures and sequence quality classified nearly all isolates with a minimum average coverage of the genome of 55x as acceptable for analysis. Assessment of the cgMLST variation between six different pipelines revealed slightly less variation associated with assembly-based analysis compared to reads-based analysis. Epidemiological concordance, based on 152 isolates from 19 confirmed outbreaks and a cluster cutoff of seven allelic differences, was good (sensitivity > 95% for two cgMLST schemes of 1,748 and 1,701 loci each; PPV 58‒68%). The proportion of sporadic cases was slightly below 50%. Of remaining isolates, around one third were in clusters involving more than one country, often spanning several years. Detection of multi-country clusters was on average several months earlier when pooling the data at EU/EEA level, compared with first detection at national level. Conclusions: These findings provide a good basis for comprehensive EU/EEA-wide, WGS-enhanced surveillance of listeriosis. Time limits should not be used for hypothesis generation during outbreak investigations, but should be for analytical studies.

Gastrointestinal microbiota contributes to the development of murine transfusion-related acute lung injury.

Transfusion-related acute lung injury (TRALI) is a syndrome of respiratory distress upon blood transfusion and is the leading cause of transfusion-related fatalities. Whether the gut microbiota plays any role in the development of TRALI is currently unknown. We observed that untreated barrier-free (BF) mice suffered from severe antibody-mediated acute lung injury, whereas the more sterile housed specific pathogen-free (SPF) mice and gut flora-depleted BF mice were both protected from lung injury. The prevention of TRALI in the SPF mice and gut flora-depleted BF mice was associated with decreased plasma macrophage inflammatory protein-2 levels as well as decreased pulmonary neutrophil accumulation. DNA sequencing of amplicons of the 16S ribosomal RNA gene revealed a varying gastrointestinal bacterial composition between BF and SPF mice. BF fecal matter transferred into SPF mice significantly restored TRALI susceptibility in SPF mice. These data reveal a link between the gut flora composition and the development of antibody-mediated TRALI in mice. Assessment of gut microbial composition may help in TRALI risk assessment before transfusion.

Peritonitis with Listeria monocytogenes in a patient on automated peritoneal dialysis.

We present a case where Listeria monocytogenesserotype 1/2a was determined to be the causative agent of peritonitis in a patient on automated peritoneal dialysis. The patient, a 53-year-old Caucasian woman from the Faroe Islands was admitted to the National Hospital reporting of constant abdominal pain and a fever. Peritoneal cultures were positive for growth of L. monocytogenes. The patient was successfully treated with oral amoxicillin for 2 weeks and intraperitoneal vancomycin for 3 weeks. To date, the patient has not been readmitted due to peritonitis. The Faroese salmon was the suspected source of infection with L. monocytogenes.

Multi-laboratory validation study of multilocus variable-number tandem repeat analysis (MLVA) for Salmonella enterica serovar Enteritidis, 2015.

Multilocus variable-number tandem repeat analysis (MLVA) is a rapid and reproducible typing method that is an important tool for investigation, as well as detection, of national and multinational outbreaks of a range of food-borne pathogens. Salmonella enterica serovar Enteritidis is the most common Salmonella serovar associated with human salmonellosis in the European Union/European Economic Area and North America. Fourteen laboratories from 13 countries in Europe and North America participated in a validation study for MLVA of S. Enteritidis targeting five loci. Following normalisation of fragment sizes using a set of reference strains, a blinded set of 24 strains with known allele sizes was analysed by each participant. The S. Enteritidis 5-loci MLVA protocol was shown to produce internationally comparable results as more than 90% of the participants reported less than 5% discrepant MLVA profiles. All 14 participating laboratories performed well, even those where experience with this typing method was limited. The raw fragment length data were consistent throughout, and the inter-laboratory validation helped to standardise the conversion of raw data to repeat numbers with at least two countries updating their internal procedures. However, differences in assigned MLVA profiles remain between well-established protocols and should be taken into account when exchanging data.

Evaluation of molecular typing of foodborne pathogens in European reference laboratories from 2012 to 2013.

In 2012, the European Centre for Disease Prevention and Control (ECDC) initiated external quality assessment (EQA) schemes for molecular typing including the National Public Health Reference Laboratories in Europe. The overall aim for these EQA schemes was to enhance the European surveillance of food-borne pathogens by evaluating and improving the quality and comparability of molecular typing. The EQAs were organised by Statens Serum Institut (SSI) and included Salmonella enterica subsp. enterica, verocytotoxin-producing Escherichia coli (VTEC) and Listeria monocytogenes. Inter-laboratory comparable pulsed-field gel electrophoresis (PFGE) images were obtained from 10 of 17 of the participating laboratories for Listeria, 15 of 25 for Salmonella, but only nine of 20 for VTEC. Most problems were related to PFGE running conditions and/or incorrect use of image acquisition. Analysis of the gels was done in good accordance with the provided guidelines. Furthermore, we assessed the multilocus variable-number tandem repeat analysis (MLVA) scheme for S. Typhimurium. Of 15 laboratories, nine submitted correct results for all analysed strains, and four had difficulties with one strain only. In conclusion, both PFGE and MLVA are prone to variation in quality, and there is therefore a continuous need for standardisation and validation of laboratory performance for molecular typing methods of food-borne pathogens in the human public health sector.

Whole genome-based population biology and epidemiological surveillance of Listeria monocytogenes.

Listeria monocytogenes (Lm) is a major human foodborne pathogen. Numerous Lm outbreaks have been reported worldwide and associated with a high case fatality rate, reinforcing the need for strongly coordinated surveillance and outbreak control. We developed a universally applicable genome-wide strain genotyping approach and investigated the population diversity of Lm using 1,696 isolates from diverse sources and geographical locations. We define, with unprecedented precision, the population structure of Lm, demonstrate the occurrence of international circulation of strains and reveal the extent of heterogeneity in virulence and stress resistance genomic features among clinical and food isolates. Using historical isolates, we show that the evolutionary rate of Lm from lineage I and lineage II is low (∼2.5 × 10-7 substitutions per site per year, as inferred from the core genome) and that major sublineages (corresponding to so-called 'epidemic clones') are estimated to be at least 50-150 years old. This work demonstrates the urgent need to monitor Lm strains at the global level and provides the unified approach needed for global harmonization of Lm genome-based typing and population biology.

Whole-genome Sequencing Used to Investigate a Nationwide Outbreak of Listeriosis Caused by Ready-to-eat Delicatessen Meat, Denmark, 2014.

BACKGROUND: Listeriosis is a serious foodborne infection. Outbreaks of listeriosis occur rarely, but have often proved difficult to solve. In June 2014, we detected and investigated a listeriosis outbreak in Denmark using patient interviews and whole-genome sequencing (WGS). METHODS: We performed WGS on Listeria monocytogenes isolates from patients and available isolates from ready-to-eat foods and compared them using single-nucleotide polymorphism (SNP) analysis. Case patients had L. monocytogenes with ≤3 SNPs (the outbreak strain) isolated in September 2013-December 2014. Through interviews, we established case patients' food and clinical histories. Food production facilities were inspected and sampled, and we performed trace-back/trace-forward of food delivery chains. RESULTS: In total, 41 cases were identified; 17 deaths occurred (41%). An isolate from a delicatessen meat (spiced meat roll) from company A was identical to the outbreak strain. Half of the patients were infected while hospitalized/institutionalized; institutions were supplied food by company A. The outbreak strain was repeatedly isolated from further samples taken within this company and within companies in its distribution chain. Products from company A were traced and recalled from >6000 food establishments, after which the outbreak ended. CONCLUSIONS: Ready-to-eat spiced meat roll from a single production facility caused this outbreak. The product, served sliced and cold, is popular among the elderly; serving it at hospitals probably contributed to the high case-fatality rate. WGS used for patient isolates and isolates from food control inspections, coupled with routine epidemiological follow-up, was instrumental in swiftly locating the source of infections, preventing further illnesses and deaths.

Molecular Typing and Epidemiology of Human Listeriosis Cases, Denmark, 2002-2012.

Denmark has a high incidence of invasive listeriosis (0.9 cases/100,000 population in 2012). We analyzed patient data, clinical outcome, and trends in pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) of Listeria monocytogenes strains isolated in Denmark during 2002-2012. We performed 2-enzyme PFGE and serotyping on 559 isolates and MLST on 92 isolates and identified some correlation between molecular type and clinical outcome and patient characteristics. We found 178 different PFGE types, but isolates from 122 cases belonged to just 2 closely related PFGE types, clonal complex 8 and sequence type 8. These 2 types were the main cause of a peak in incidence of invasive listeriosis during 2005-2009, possibly representing an outbreak or the presence of a highly prevalent clone. However, current typing methods could not fully confirm these possibilities, highlighting the need for more refined discriminatory typing methods to identify outbreaks within frequently occurring L. monocytogenes PFGE types.

Failure of microtubule-mediated peroxisome division and trafficking in disorders with reduced peroxisome abundance.

In contrast to peroxisomes in normal cells, remnant peroxisomes in cultured skin fibroblasts from a subset of the clinically severe peroxisomal disorders that includes the biogenesis disorder Zellweger syndrome and the single-enzyme defect D-bifunctional protein (D-BP) deficiency, are enlarged and significantly less abundant. We tested whether these features could be related to the known role of microtubules in peroxisome trafficking in mammalian cells. We found that remnant peroxisomes in fibroblasts from patients with PEX1-null Zellweger syndrome or D-BP deficiency exhibited clustering and loss of alignment along peripheral microtubules. Similar effects were observed for both cultured embryonic fibroblasts and brain neurons from a PEX13-null mouse with a Zellweger-syndrome-like phenotype, and a less-pronounced effect was observed for fibroblasts from an infantile Refsum patient who was homozygous for a milder PEX1 mutation. By contrast, such changes were not seen for patients with peroxisomal disorders characterized by normal peroxisome abundance and size. Stable overexpression of PEX11beta to induce peroxisome proliferation largely re-established the alignment of peroxisomal structures along peripheral microtubules in both PEX1-null and D-BP-deficient cells. In D-BP-deficient cells, peroxisome division was apparently driven to completion, as induced peroxisomal structures were similar to the spherical parental structures. By contrast, in PEX1-null cells the majority of induced peroxisomal structures were elongated and tubular. These structures were apparently blocked at the division step, despite having recruited DLP1, a protein necessary for peroxisome fission. These findings indicate that the increased size, reduced abundance, and disturbed cytoplasmic distribution of peroxisomal structures in PEX1-null and D-BP-deficient cells reflect defects at different stages in peroxisome proliferation and division, processes that require association of these structures with, and dispersal along, microtubules.

Pex13 inactivation in the mouse disrupts peroxisome biogenesis and leads to a Zellweger syndrome phenotype.

Zellweger syndrome is the archetypical peroxisome biogenesis disorder and is characterized by defective import of proteins into the peroxisome, leading to peroxisomal metabolic dysfunction and widespread tissue pathology. In humans, mutations in the PEX13 gene, which encodes a peroxisomal membrane protein necessary for peroxisomal protein import, can lead to a Zellweger phenotype. To develop mouse models for this disorder, we have generated a targeted mouse with a loxP-modified Pex13 gene to enable conditional Cre recombinase-mediated inactivation of Pex13. In the studies reported here, we crossed these mice with transgenic mice that express Cre recombinase in all cells to generate progeny with ubiquitous disruption of Pex13. The mutant pups exhibited many of the clinical features of Zellweger syndrome patients, including intrauterine growth retardation, severe hypotonia, failure to feed, and neonatal death. These animals lacked morphologically intact peroxisomes and showed deficient import of matrix proteins containing either type 1 or type 2 targeting signals. Biochemical analyses of tissue and cultured skin fibroblasts from these animals indicated severe impairment of peroxisomal fatty acid oxidation and plasmalogen synthesis. The brains of these animals showed disordered lamination in the cerebral cortex, consistent with a neuronal migration defect. Thus, Pex13(-/-) mice reproduce many of the features of Zellweger syndrome and PEX13 deficiency in humans.

Pex13, the mouse ortholog of the human peroxisome biogenesis disorder PEX13 gene: gene structure, tissue expression, and localization of the protein to peroxisomes.

Pex13 encodes an SH3-containing peroxisomal membrane protein required for the import of proteins into peroxisomes. In humans, mutations in PEX13 can disrupt peroxisome biogenesis and lead to peroxisomal metabolic dysfunction and neurodegenerative disease. We report here on the mouse gene Pex13 and its encoded protein. Mouse Pex13 spans 18 kb and consists of four exons. We detected Pex13 transcripts in all mouse tissues tested, with highest levels in liver and testis. The Pex13 open reading frame predicts a 44.5-kDa protein that displays 91% sequence identity to the human PEX13 protein. We have localized PEX13 protein to peroxisomes in mouse liver and show that this protein also sorts to peroxisomes in human skin fibroblasts. These data indicate that the structure and properties of the mouse and human PEX13 proteins are almost identical. We infer from these findings that targeted disruption of mouse Pex13 would provide an appropriate model for the study of PEX13 dysfunction in humans.