Guideline: Use of whole genome sequencing for prevention and control of Listeria monocytogenes in the food industry

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Metoder for påvisning av Listeria i mat og produksjonsmiljø – Delrapport 3: Genotypingsmetoder

Sporing av Listeria monocytogenes er en viktig del av lakseprodusenters kvalitetskontroll. Det er økende interesse i laksenæringen for metoder som kan gi mer informasjon enn bare et svar på om listeria er til stede i en prøve eller ikke – altså genotypingsmetoder. Denne rapporten gir en grunnleggende oversikt over prinsippene bak eksisterende genotypingsmetoder, inkludert PCR-basert typing, MLST, MLVA og PFGE. Rapporten gir også en evaluering av metodenes oppløselighet når det gjelder å skille mellom relevante genetiske grupper av listeria, som er kjent gjennom analyser av helgenomsekvenseringdata. En viktig del av rapporten er en evaluering av den nye GENE-UP Typer metoden fra bioMérieux, utført som en digital analyse av et datasett bestående av over 1200 norske listeria-isolater. Resultatene viste at GENE-UP Typer metoden hadde noen fordeler sammenlignet med de klassiske genotypingsmetodene, inkludert et lavt nivå av feilklassifiseringer, men ikke høyere oppløselighet enn andre metoder. Genotyping kan være et nyttig verktøy i kvalitetsarbeidet, men kan ikke erstatte helgenomsekvensering for å spore smitte i næringsmiddelkjeden eller i produksjonsmiljøet i bedrifter.publishedVersio

Metoder for påvisning av Listeria i mat og produksjonsmiljø – Delrapport 2: Nye metoder og teknologier

Det er et stort ønske fra aktører i sjømatindustrien å få en raskere metode for påvisning av listeria. Som en del av et FHF prosjekt så er det vurdert om det er faglig og teknisk mulig å utvikle en metode for påvisning av listeria i løpet av 20-30 minutter. Selv om det er framskritt på forskningsfronten på hurtigmetoder så er det fortsatt mangler på enten sensitivitet, selektivitet eller robusthet. Med dagens regelverk og teknologi vil det ikke være mulig å utvikle en hurtigtest som tar 20-30 minutter. Det er likevel rom for forbedring av dagens metoder i form av kortere og mer selektiv oppformering, mer presise metoder (f.eks. skille mellom levende og døde bakterier), samt få mer informasjon i form av typing. Det kan kanskje være mulig å oppnå en metode som tar 8-10 timer, noe som ville være veldig nyttig for sjømatindustrien. Rapporten inneholder en oppsummering av en brukerundersøkelse, og en oversikt over nye metoder fra vitenskapelige publikasjoner.Metoder for påvisning av Listeria i mat og produksjonsmiljø – Delrapport 2: Nye metoder og teknologierpublishedVersio

Metoder for påvisning av Listeria i mat og produksjonsmiljø – Faglig sluttrapport

Dagens referansemetode for påvisning av Listeria monocytogenes tar minimum to dager før man får et endelig svar. Dette reduserer mulighetene for å gjøre risikoreduserende tiltak. Prosjektet har kartlagt dagens kommersielle metoder og gitt en oversikt over nye teknologier og metoder for å vurdere om det er teknologisk mulig å få påvisningstiden ned til 20-30 minutter. Dagens raskeste validerte metode (real-time PCR) tar 20 timer hvorav 90% av tiden er oppformeringstrinnet og 10% er påvisningstrinnet. Dersom teknologiske barrierer overkommes og markedet etterspør det, kan det i framtiden være mulig å komme ned på en analysetid på 8-10 timer. Dette vil bety at en stor andel laks- og ørretbedrifter kan varsle renholdere før neste renhold og holde tilbake produkter før de når markedet. Det kan være mulig å utvikle metodikk med kortere tid før analysesvar, i beste fall ned mot 30 minutter, men den vil være mindre sensitiv enn regelverket krever. Denne typen tester vil likevel kunne være et nyttig tillegg til ISO-validerte metoder. Nye metoder bør i tillegg til hurtighet fokusere på å skille mellom levende og døde bakterier, samt gi mer enn bare et positivt eller negativt svar, dvs. genotyping.Metoder for påvisning av Listeria i mat og produksjonsmiljø – Faglig sluttrapportpublishedVersio

Metoder for påvisning av Listeria i mat og produksjonsmiljø – Delrapport 1: Hurtigmetoder på markedet

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Toxin production in a rare and genetically remote cluster of strains of the Bacillus cereus group

<p>Abstract</p> <p>Background</p> <p>Three enterotoxins are implicated in diarrhoeal food poisoning due to <it>Bacillus cereus</it>: Haemolysin BL (Hbl), Non-haemolytic enterotoxin (Nhe), and Cytotoxin K (CytK). Toxin gene profiling and assays for detection of toxin-producing stains have been used in attempts to evaluate the enterotoxic potential of <it>B. cereus </it>group strains. <it>B. cereus </it>strain NVH 391/98, isolated from a case of fatal enteritis, was genetically remote from other <it>B. cereus </it>group strains. This strain lacked the genes encoding Hbl and Nhe, but contains CytK-1. The high virulence of this strain is thought to be due to the greater cytotoxic activity of CytK-1 compared to CytK-2, and to a high level of <it>cytK </it>expression. To date, only three strains containing <it>cytK-1 </it>have been identified; <it>B. cereus </it>strains NVH 391/98, NVH 883/00, and INRA AF2.</p> <p>Results</p> <p>A novel gene variant encoding Nhe was identified in these three strains, which had an average of 80% identity in protein sequence with previously identified Nhe toxins. While culture supernatants containing CytK and Nhe from NVH 391/98 and INRA AF2 were highly cytotoxic, NVH 883/00 expressed little or no CytK and Nhe and was non-cytotoxic. Comparative sequence and expression studies indicated that neither the PlcR/PapR quorum sensing system, nor theYvrGH and YvfTU two-component systems, were responsible for the observed difference in toxin production. Additionally, phylogenetic analysis of 13 genes showed that NVH 391/98, NVH 883/00, and INRA AF2 comprise a novel cluster of strains genetically distant from other <it>B. cereus </it>group strains.</p> <p>Conclusion</p> <p>Due to its divergent sequence, the novel <it>nhe </it>operon had previously not been detected in NVH 391/98 using PCR and several monoclonal antibodies. Thus, toxigenic profiling based on the original <it>nhe </it>sequence will fail to detect the toxin in this group of strains. The observation that strain NVH 883/00 carries <it>cytK-1 </it>but is non-cytotoxic indicates that the detection of this gene variant is not a sufficient criterion for identification of highly cytotoxic strains. The presence of the novel <it>nhe </it>operon and the <it>cytK-1 </it>gene variant in this cluster of strains reflect their phylogenetically remote relationship towards other <it>B. cereus </it>group strains.</p

Pervasive Listeria monocytogenes is common in the Norwegian food system and is associated with increased prevalence of stress survival and resistance determinants

To investigate the diversity, distribution, persistence, and prevalence of stress survival and resistance genes of Listeria monocytogenes clones dominating in food processing environments in Norway, genome sequences from 769 L. monocytogenes isolates from food industry environments, foods, and raw materials (512 of which were sequenced in the present study) were subjected to whole-genome multilocus sequence typing (wgMLST), single-nucleotide polymorphism (SNP), and comparative genomic analyses. The data set comprised isolates from nine meat and six salmon processing facilities in Norway collected over a period of three decades. The most prevalent clonal complex (CC) was CC121, found in 10 factories, followed by CC7, CC8, and CC9, found in 7 factories each. Overall, 72% of the isolates were classified as persistent, showing 20 or fewer wgMLST allelic differences toward an isolate found in the same factory in a different calendar year. Moreover, over half of the isolates (56%) showed this level of genetic similarity toward an isolate collected from a different food processing facility. These were designated as pervasive strains, defined as clusters with the same level of genetic similarity as persistent strains but isolated from different factories. The prevalence of genetic determinants associated with increased survival in food processing environments, including heavy metal and biocide resistance determinants, stress response genes, and inlA truncation mutations, showed a highly significant increase among pervasive isolates but not among persistent isolates. Furthermore, these genes were significantly more prevalent among the isolates from food processing environments compared to in isolates from natural and rural environments (n = 218) and clinical isolates (n = 111) from NorwaypublishedVersio

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

Listeria monocytogenes is a ubiquitous environmental bacterium associated with a wide variety of natural and human-made environments, such as soil, vegetation, livestock, food processing environments, and urban areas. It is also among the deadliest foodborne pathogens, and knowledge about its presence and diversity in potential sources is crucial to effectively track and control it in the food chain. Isolation of L. monocytogenes from various rural and urban environments showed higher prevalence in agricultural and urban developments than in forest or mountain areas, and that detection was positively associated with rainfall. Whole-genome sequencing (WGS) was performed for the collected isolates and for L. monocytogenes from Norwegian dairy farms and slugs (218 isolates in total). The data were compared to available data sets from clinical and food-associated sources in Norway collected within the last decade. Multiple examples of clusters of isolates with 0 to 8 whole-genome multilocus sequence typing (wgMLST) allelic differences were collected over time in the same location, demonstrating persistence of L. monocytogenes in natural, urban, and farm environments. Furthermore, several clusters with 6 to 20 wgMLST allelic differences containing isolates collected across different locations, times, and habitats were identified, including nine clusters harboring clinical isolates. The most ubiquitous clones found in soil and other natural and animal ecosystems (CC91, CC11, and CC37) were distinct from clones predominating among both clinical (CC7, CC121, and CC1) and food (CC9, CC121, CC7, and CC8) isolates. The analyses indicated that ST91 was more prevalent in Norway than other countries and revealed a high proportion of the hypovirulent ST121 among Norwegian clinical cases.publishedVersio

Deciphering the virulence potential of Listeria monocytogenes in the Norwegian meat and salmon processing industry by combining whole genome sequencing and in vitro data

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Listeria Monocytogenes Biofilm Removal Using Different Commercial Cleaning Agents

Effective cleaning and disinfection (C&amp;D) is pivotal for the control of Listeria monocytogenes in food processing environments. Bacteria in biofilms are protected from biocidal action, and effective strategies for the prevention and removal of biofilms are needed. In this study, different C&amp;D biofilm control strategies on pre-formed L. monocytogenes biofilms on a conveyor belt material were evaluated and compared to the effect of a conventional chlorinated, alkaline cleaner (agent A). Bacterial reductions up to 1.8 log were obtained in biofilms exposed to daily C&amp;D cycles with normal user concentrations of alkaline, acidic, or enzymatic cleaning agents, followed by disinfection using peracetic acid. No significant differences in bactericidal effects between the treatments were observed. Seven-day-old biofilms were more tolerant to C&amp;D than four-day-old biofilms. Attempts to optimize biofilm eradication protocols for four alkaline, two acidic, and one enzymatic cleaning agent, in accordance with the manufacturers&rsquo; recommendations, were evaluated. Increased concentrations, the number of subsequent treatments, the exposure times, and the temperatures of the C&amp;D agents provided between 4.0 and &gt;5.5 log reductions in colony forming units (CFU) for seven-day-old L. monocytogenes biofilms. Enhanced protocols of conventional and enzymatic C&amp;D protocols have the potential for improved biofilm control, although further optimizations and evaluations are needed