Large Multicountry Outbreak of Invasive Listeriosis by a Listeria monocytogenes ST394 Clone Linked to Smoked Rainbow Trout, 2020 to 2021

Listeriosis is a severe infectious disease in humans and characterized by an exceptionally high case fatality rate. The disease is transmitted through consumption of food contaminated by the bacterium Listeria monocytogenes. ABSTRACT Whole-genome sequencing (WGS) has revolutionized surveillance of infectious diseases. Disease outbreaks can now be detected with high precision, and correct attribution of infection sources has been improved. Listeriosis, caused by the bacterium Listeria monocytogenes, is a foodborne disease with a high case fatality rate and a large proportion of outbreak-related cases. Timely recognition of listeriosis outbreaks and precise allocation of food sources are important to prevent further infections and to promote public health. We report the WGS-based identification of a large multinational listeriosis outbreak with 55 cases that affected Germany, Austria, Denmark, and Switzerland during 2020 and 2021. Clinical isolates formed a highly clonal cluster (called Ny9) based on core genome multilocus sequence typing (cgMLST). Routine and ad hoc investigations of food samples identified L. monocytogenes isolates from smoked rainbow trout filets from a Danish producer grouping with the Ny9 cluster. Patient interviews confirmed consumption of rainbow trout as the most likely infection source. The Ny9 cluster was caused by a MLST sequence type (ST) ST394 clone belonging to molecular serogroup IIa, forming a distinct clade within molecular serogroup IIa strains. Analysis of the Ny9 genome revealed clpY, dgcB, and recQ inactivating mutations, but phenotypic characterization of several virulence-associated traits of a representative Ny9 isolate showed that the outbreak strain had the same pathogenic potential as other serogroup IIa strains. Our report demonstrates that international food trade can cause multicountry outbreaks that necessitate cross-border outbreak collaboration. It also corroborates the relevance of ready-to-eat smoked fish products as causes for listeriosis. IMPORTANCE Listeriosis is a severe infectious disease in humans and characterized by an exceptionally high case fatality rate. The disease is transmitted through consumption of food contaminated by the bacterium Listeria monocytogenes. Outbreaks of listeriosis often occur but can be recognized and stopped through implementation of whole-genome sequencing-based pathogen surveillance systems. We here describe the detection and management of a large listeriosis outbreak in Germany and three neighboring countries. This outbreak was caused by rainbow trout filet, which was contaminated by a L. monocytogenes clone belonging to sequence type ST394. This work further expands our knowledge on the genetic diversity and transmission routes of an important foodborne pathogen.

[1]  S. Halbedel,et al.  Listeria monocytogenes genes supporting growth under standard laboratory cultivation conditions and during macrophage infection , 2022, Genome research.

[2]  Ariane T. Pietzka,et al.  Invasive listeriosis outbreaks and salmon products: a genomic, epidemiological study , 2022, Emerging microbes & infections.

[3]  S. Brisse,et al.  Is it the same strain? Defining genomic epidemiology thresholds tailored to individual outbreaks , 2022, bioRxiv.

[4]  T. Møretrø,et al.  Whole-Genome Sequencing Analysis of Listeria monocytogenes from Rural, Urban, and Farm Environments in Norway: Genetic Diversity, Persistence, and Relation to Clinical and Food Isolates , 2022, Applied and environmental microbiology.

[5]  The European Union One Health 2020 Zoonoses Report , 2021, EFSA journal. European Food Safety Authority.

[6]  Maxime Pichon,et al.  Listeria monocytogenes faecal carriage is common and depends on the gut microbiota , 2021, Nature Communications.

[7]  A. Flieger,et al.  Ongoing High Incidence and Case-Fatality Rates for Invasive Listeriosis, Germany, 2010–2019 , 2021, Emerging infectious diseases.

[8]  A. Flieger,et al.  Complete Genome Sequences of Three Clinical Listeria monocytogenes Sequence Type 8 Strains from Recent German Listeriosis Outbreaks , 2021, Microbiology Resource Announcements.

[9]  P. Bork,et al.  Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation , 2021, Nucleic Acids Res..

[10]  J. Quereda,et al.  Pathogenicity and virulence of Listeria monocytogenes: A trip from environmental to medical microbiology , 2021, Virulence.

[11]  F. Allerberger,et al.  Nationwide outbreak of invasive listeriosis associated with consumption of meat products in health care facilities, Germany, 2014-2019. , 2020, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[12]  G. Bange,et al.  Physiology of guanosine-based second messenger signaling in Bacillus subtilis , 2020, Biological chemistry.

[13]  S. Huhulescu,et al.  Large Nationwide Outbreak of Invasive Listeriosis Associated with Blood Sausage, Germany, 2018–2019 , 2020, Emerging infectious diseases.

[14]  Juno Thomas,et al.  Outbreak of Listeriosis in South Africa Associated with Processed Meat. , 2020, The New England journal of medicine.

[15]  A. Flieger,et al.  Backtracking and forward checking of human listeriosis clusters identified a multiclonal outbreak linked to Listeria monocytogenes in meat products of a single producer , 2020, Emerging microbes & infections.

[16]  F. Allerberger,et al.  Population structure-guided profiling of antibiotic resistance patterns in clinical Listeria monocytogenes isolates from Germany identifies pbpB3 alleles associated with low levels of cephalosporin resistance , 2020, bioRxiv.

[17]  S. Huhulescu,et al.  Whole Genome Sequencing Based Surveillance of L. monocytogenes for Early Detection and Investigations of Listeriosis Outbreaks , 2019, Front. Public Health.

[18]  T. Dallman,et al.  LiSEQ – whole-genome sequencing of a cross-sectional survey of Listeria monocytogenes in ready-to-eat foods and human clinical cases in Europe , 2019, Microbial genomics.

[19]  E. Trost,et al.  A Listeria monocytogenes ST2 clone lacking chitinase ChiB from an outbreak of non-invasive gastroenteritis , 2019, Emerging microbes & infections.

[20]  Multi‐country outbreak of Listeria monocytogenes serogroup IVb, multi‐locus sequence type 6, infections linked to frozen corn and possibly to other frozen vegetables – first update , 2018, EFSA Supporting Publications.

[21]  Stephan Fuchs,et al.  Whole-Genome Sequencing of Recent Listeria monocytogenes Isolates from Germany Reveals Population Structure and Disease Clusters , 2018, Journal of Clinical Microbiology.

[22]  Yun Chen,et al.  The ClpY-ClpQ protease regulates multicellular development in Bacillus subtilis. , 2018, Microbiology.

[23]  S. Fuchs,et al.  Whole Genome Sequence Analysis of CTX-M-15 Producing Klebsiella Isolates Allowed Dissecting a Polyclonal Outbreak Scenario , 2018, Front. Microbiol..

[24]  Alexandre Leclercq,et al.  Cross-border outbreak of listeriosis caused by cold-smoked salmon, revealed by integrated surveillance and whole genome sequencing (WGS), Denmark and France, 2015 to 2017 , 2017, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[25]  S. Brisse,et al.  Real-Time Whole-Genome Sequencing for Surveillance of Listeria monocytogenes, France , 2017, Emerging infectious diseases.

[26]  Dereje D. Jima,et al.  The Arsenic Resistance-Associated Listeria Genomic Island LGI2 Exhibits Sequence and Integration Site Diversity and a Propensity for Three Listeria monocytogenes Clones with Enhanced Virulence , 2017, Applied and Environmental Microbiology.

[27]  E. Charpentier,et al.  Functional Diversity of AAA+ Protease Complexes in Bacillus subtilis , 2017, Front. Mol. Biosci..

[28]  M. Wagner,et al.  Stress Survival Islet 2, Predominantly Present in Listeria monocytogenes Strains of Sequence Type 121, Is Involved in the Alkaline and Oxidative Stress Responses , 2017, Applied and Environmental Microbiology.

[29]  P. Ravaud,et al.  Clinical features and prognostic factors of listeriosis: the MONALISA national prospective cohort study. , 2017, The Lancet. Infectious diseases.

[30]  Yun Chen,et al.  Genome-Wide Investigation of Biofilm Formation in Bacillus cereus , 2017, Applied and Environmental Microbiology.

[31]  Ryan R. Wick,et al.  Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads , 2016, bioRxiv.

[32]  Eduardo P C Rocha,et al.  Whole genome-based population biology and epidemiological surveillance of Listeria monocytogenes , 2016, Nature Microbiology.

[33]  Yi Chen,et al.  Implementation of Nationwide Real-time Whole-genome Sequencing to Enhance Listeriosis Outbreak Detection and Investigation. , 2016, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[34]  K. Mølbak,et al.  Two listeria outbreaks caused by smoked fish consumption-using whole-genome sequencing for outbreak investigations. , 2016, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[35]  Eric P. Nawrocki,et al.  NCBI prokaryotic genome annotation pipeline , 2016, Nucleic acids research.

[36]  David S. Wishart,et al.  PHASTER: a better, faster version of the PHAST phage search tool , 2016, Nucleic Acids Res..

[37]  Steen Ethelberg,et al.  Whole-genome Sequencing Used to Investigate a Nationwide Outbreak of Listeriosis Caused by Ready-to-eat Delicatessen Meat, Denmark, 2014. , 2016, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[38]  Eduardo P C Rocha,et al.  Uncovering Listeria monocytogenes hypervirulence by harnessing its biodiversity , 2016, Nature Genetics.

[39]  S. Huhulescu,et al.  Ongoing outbreak of invasive listeriosis, Germany, 2012 to 2015. , 2015, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[40]  M. Gilmour,et al.  Tolerance of Listeria monocytogenes to Quaternary Ammonium Sanitizers Is Mediated by a Novel Efflux Pump Encoded by emrE , 2015, Applied and Environmental Microbiology.

[41]  Dag Harmsen,et al.  Defining and Evaluating a Core Genome Multilocus Sequence Typing Scheme for Whole-Genome Sequence-Based Typing of Listeria monocytogenes , 2015, Journal of Clinical Microbiology.

[42]  Parastoo Azadi,et al.  Listeria monocytogenes exopolysaccharide: origin, structure, biosynthetic machinery and c‐di‐GMP‐dependent regulation , 2015, Molecular microbiology.

[43]  N. Speybroeck,et al.  The global burden of listeriosis: a systematic review and meta-analysis. , 2014, The Lancet. Infectious diseases.

[44]  J. Vendôme,et al.  Characterization of Biochemical Properties of Bacillus subtilis RecQ Helicase , 2014, Journal of bacteriology.

[45]  Mark Gomelsky,et al.  Cyclic di-GMP-dependent Signaling Pathways in the Pathogenic Firmicute Listeria monocytogenes , 2014, PLoS pathogens.

[46]  S. Engelmann,et al.  A Systematic Proteomic Analysis of Listeria monocytogenes House-keeping Protein Secretion Systems* , 2014, Molecular & Cellular Proteomics.

[47]  R. Evans.  European Centre for Disease Prevention and Control. , 2014, Nursing standard (Royal College of Nursing (Great Britain) : 1987).

[48]  D. Garmyn,et al.  Listeria monocytogenes, a down-to-earth pathogen , 2013, Front. Cell. Infect. Microbiol..

[49]  J. R. Gorny,et al.  Multistate outbreak of listeriosis associated with cantaloupe. , 2013, The New England journal of medicine.

[50]  V. Vaillant,et al.  What is the incubation period for listeriosis? , 2013, BMC Infectious Diseases.

[51]  J. Müller-Nordhorn,et al.  Years of potential life lost for six major enteric pathogens, Germany, 2004–2008 , 2012, Epidemiology and Infection.

[52]  K. G. Kaval,et al.  Architecturally the same, but playing a different game: the diverse species-specific roles of DivIVA proteins. , 2012, Virulence.

[53]  K. Volz,et al.  Probing the Role of Protein Surface Charge in the Activation of PrfA, the Central Regulator of Listeria monocytogenes Pathogenesis , 2011, PloS one.

[54]  C. Gahan,et al.  A five‐gene stress survival islet (SSI‐1) that contributes to the growth of Listeria monocytogenes in suboptimal conditions , 2010, Journal of applied microbiology.

[55]  S. Huhulescu,et al.  Listeriosis outbreak caused by acid curd cheese Quargel , Austria and Germany 2009. , 2010, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[56]  M. Loessner,et al.  Comparative Genome Analysis of Listeria Bacteriophages Reveals Extensive Mosaicism, Programmed Translational Frameshifting, and a Novel Prophage Insertion Site , 2009, Journal of bacteriology.

[57]  S. Brisse,et al.  A New Perspective on Listeria monocytogenes Evolution , 2008, PLoS pathogens.

[58]  Pascale Cossart,et al.  Identification of new noncoding RNAs in Listeria monocytogenes and prediction of mRNA targets , 2007, Nucleic acids research.

[59]  Patricia Siguier,et al.  ISfinder: the reference centre for bacterial insertion sequences , 2005, Nucleic Acids Res..

[60]  David S. Wishart,et al.  Circular genome visualization and exploration using CGView , 2005, Bioinform..

[61]  W. Heyer.  Damage Signaling: RecQ Sends an SOS to You , 2004, Current Biology.

[62]  F. Allerberger,et al.  Incidence of Fecal Carriage of Listeria monocytogenes in Three Healthy Volunteers: A One-Year Prospective Stool Survey , 2003, European Journal of Clinical Microbiology and Infectious Diseases.

[63]  L. Gautier,et al.  Comparative Genomics of Listeria Species , 2001, Science.

[64]  S. Salmaso,et al.  An outbreak of febrile gastroenteritis associated with corn contaminated by Listeria monocytogenes. , 2000, The New England journal of medicine.

[65]  L. Domínguez,et al.  Taxonomic note: a proposal for reviewing the interpretation of the CAMP reaction between Listeria monocytogenes and Rhodococcus equi. , 1996, International journal of systematic bacteriology.

[66]  R. Appleyard.  Segregation of Lambda Lysogenicity during Bacterial Recombination in Escherichia Coli K12. , 1954, Genetics.