Existing Seafood Pathogens and Future Seafood Safety Concerns

1. Existing Seafood Pathogens and Future Seafood Safety Concerns Taran Skjerdal taran.skjerdal@vetinst.no tlf +4723216268 AOAC meeting: Setting Performance Requirements for New and Modern Methods Inaugural meeting, June 25-26 2009, Rockville, USA

2. Objectives of the talk:Give an introduction to • Special concerns with seafood • Global trade • Water • Pathogens in seafood from different geographic areas • Effect of water temperature, farming conditions and seafood matrix • Effects of processing on food safety and quality • Trends in legislation • Differentiated limit values at various stages in the farm-to-fork chain • Focus on ready-to-eat products

3. Seafood is a global trade Existing and upcoming (expected) production areas, as it was seen in 2007

4. 0-3 days +1-2 days +1-3 days +1-7.. days Fish has a short shelf life, But goes through many Steps along the farm-to-fork chain – where microbes can enter and multiply and even change

5. Processing to ready-to-eat products, often in transit countries, is more common than before Photo: T Skjerdal

6. Fish products with various risk levelsRaw – lightly preserved – undercooked and stored – cross contaminated

7. Traditional analysis method in trade: Sensory quality assessment during storage Torry scale - QIM (Quality index method) Ideal conditions Sensory quality (QIM-score) deviations due to catch damages etc Microbial spoilage Storage on ice (days) Photo: T Skjerdal A scoring system for sensory attributes Linear correlation with storage time A good and rapid system for quality and freshness assessment – when the head is on and inspection is possible Read more on: http://www.qim-eurofish.com/default.asp?ZNT=S0T1O13

8. In large scale production and distribution….. …inspection is not always possible or cost effective. At the German border: The procedure for opening a container takes 8 hours. Photo: T Skjerdal …the fish head is not always on. Other analysis methods are needed for quality and safety aspects

9. Analysis needed: • Assessment and control of food safety of raw materials and products • Assess legal aspects and trustworthyness in international trade when • traceability data are diffuse or missing • the suppliers are new or not known • the risk agents may vary • Analysis methods should preferably be • Possible at the time when decisions have to be taken • Cost effective • Possible to combine with predictive models, meaning that analysis at one step in the farm-to-fork chain should make it possible to estimate the food safety risk later in the chain. Consequence: measurement of total viable count may in some cases give more information than the presence/absence of a specific pathogen. • Quantitative-Detection is in many cases not enough – the risk is often more related to level

10. Microbes grow Microbes grow very slowly, but enough to balance for those who die off – waters with high content of organic material but low temperature Microbes die off – waters with low temperature and/or low content of organic material microbe level in the water Time Presence of microbes in sea water depends on for instance water temperature, contamination level, etc • Case: A farming locality is continuously supplied with human pathogenic bacteria from the fish feed (Salmonella) humans and mammalian sewage (Listeria, Vibrio, Shigella, viruses). • Which strains will survive in 1) cold water and 2) temperate waters?

11. Lower treshold temp Reservoire for growth Infective dose (example) L. monocytogenes: 2-4 °C 10?-108 cfu Everywhere Vibrio parahaemolyticus : above 8°C 108 cfu water, sediments Salmonella spp: 8 °C, 1- 102, 103-106 cfu birds, etc Shigella spp: 7 °C 10 -105 cfu Humans Histamine forming bacteria 0 - ? 105 (estimated) Sea water, skin, process eqp. Staphylococcus aureus (toxine production) app 12 °C more than 105 cfu/g Everywhere, mainly humans Viruses (human pathogen) ? app 106 Humans, water • The water temperature varies between areas – ”living seafood” has the same temperature as the surrounding water. • The temperature in fish counters is higher than the threshold value for growth in many countries

12. Some pathogens are more relevant in some areas than others: • The threshold temperature for growth of specific pathogens explains why seafood from cold water areas contain lower levels and fewer pathogens than seafood from temperate water areas, even if both areas are in polluted areas. • Example: Human pathogens Vibrio spp and Salmonella spp are hardly present in cold water areas, but represent food safety risks in warmer areas. • Emerging pathogens are not only due to increased travelling, production hygiene and pollution, but also to changed temperatures/climate.

13. Pathogens in mussels and oysters • Filter large amounts of water, accumulate pathogens in the water in the muscle, i.e. the part we eat. • Oysters are eaten raw, and are therefore high risk products Improve food safety: • Monitoring: Analysis of sea water to detect pathogens and algal blooms, etc • Avoid muscle production/farming in polluted areas to reduse the risk of contamination from humans.

14. Fresh fish is not on the list of risk products, but traces of pathogens, including L. monocytogenes may be present on Gills Skin Sea water On equipment In slaughter houses In processing plants and grow to infective doses during processing, storage and distribution Photo: T Skjerdal

15. By slaughtering and primary processing After distribution and secondary processing at abuse temperature at ideal temperature 1 2 3 4 5 6 7 Human pathogenic bacteria level in fish (log cfu/g) ”Distribution” of levels of human pathogenic microbes early and late in the farm-to-fork chain(illustration, not based on real data) Abuse temperature in this example: Temperature above threshold value for growth The lower threshold temperature for growth, the higher risk of high levels in the product

16. Will a fish with high levels of pathogens be consumed, or sorted out due to spoilage? 100000000 1000000 10000 100 1 Spoilage bacteria Unsafe product Sensoryspoiled CFU/g fish Human pathogen 0 2 4 6 8 10 12 Days at 5-8°C Raw fish becomes spoiled before it is unsafe to eat (raw) – as long as the consumers are able to recognise spoilage Preserved products may become unsafe before spoiled if human pathogenic bacteria are present in low levels Preservation may be a sieve where pathogens can go through

17. Example: Histamine poisoning due to preserved tuna • Histamine production is a problem only in fish that contains high levels of histidine, the precursor for histamine. Species: tuna, sword fish, mackerel, (herring), etc. • Similar for other biogenic amines: • Histamine is formed both in chemical and microbial reactions. • Cold adapted histamine producing bacteria: • Morganella psychrotolerans (identified by Emborg og Dalgaard, Journal of Food Protection 2006) • Photobacterium phosphoreum, present in most fish species • Spoilage indicator for tuna: loss of fresh red colour. But: the colour can be maintained during storage by use of preservatives. Therefore, histamine can be formed even if the fish look fresh. • Analysis of histamine (Based on Dalgaard, 2007): • The level is highest near the belly flaps, lowest in the tail part of the fillets. • The level of histamine is nearly always higher than for other biogenic amines

18. Is there any risk that a native fillet may cause a food safety risk before the fish is spoiled? Spoilage of fresh fish • Specific spoilage bacteria in fish (for further reading, see review papers of Gram, Dalgaard and Huus, International journal of Food Microbiology, 1993 and 1996) • Sulphide producing bacteria; mainly Shewanella putrefaciens. • Grows in air and vacuum, but not in MAP • TMA forming bacteria; Photobacterium phosphoreum and Shewanella putrefaciens • P. phosphoreum grows in air, vacuum and MAP • Both kinds: • Present in sea water, skin mucous, etc • Grows even during ice storage.

19. Growth rate of sulphide producing bacteria in fish Max 24 doubling times before most consumers consider the fish spoiled Doubling time depends on temperature: Temperature Doubling time 24 doubling times 0 °C: ca 15 hours ca 15 days 4 ” ca 6 ” ca 6 ” 7 ” ca 4 ” ca 4 ” 10 ” ca 2,5 ” ca 2,5 ” 20 ” max 1 ” max 1 ” Based on data from own experiments and data from Fiskeriforskning and DIFRES. 1 2 4 8 16 32 64 128 Ca 250 500 1000 2000 4000 8000 16000 32000 64000 128000 Ca 250000 500000 1000000 2000000 4000000 8000000 16000000 32000000 64000000 128000000 10 millions SPB per gram fish makes it smell putride/spoiled

20. 0 °C No growth observed 4 °C 8 °C 12 °C 0 1 2 3 4 5 6 7 8 Days Temperatures above threshold for growthTime for 100 fold doubling of Listeria spp in inoculated salmon, including the lag period. But, as fish spoils rapidly, will fish stored at such conditions be sold to consumers?

21. Risk of Listeria monocytogenes infection depends on: • Amount of Listeria monocytogenes in food consumed • A fraction of the bacterium survives through stomach acid conditions • Ability to cause invasive infection and illness

22. Sushi study:Which fish quality is sold for preparation of sushi at home – can the Listeria dose reach 100 cfu/g? Shopping study: • Person 1: Ordered fish without asking questions • Person 2: Asked for fish suited for preparation of sushi for a 40 year birthday with one pregnant guest. • Visited 19 shops Results • Same quality offered for all kinds of dishes • Poor knowledge about food safety among the staff • 5 % of the samples appeared to be stored at conditions allowing a 100 fold increase of L. monocytogenes Foto: Taran Skjerdal

23. Estimated the time-temperture history:Combine analyses with traceability data, and calculate quality and safety information. Stored at abuse Temperature, contaminated during production or older than informed Quality degrading parameter (log SPB) Stored on ice! Storage time (days) Traceability data: Catch/Slaughter date predictive model of a quality parameter related to time and temperature, test method for verification of predicted quality parameter value Developed by T. Skjerdal during work in DNV

24. Analysis methods for sulphide producing bacteria applied: NMKL 96 og FAST Sulfidproduserende bakterier i islagret torsk 6 5 Predicted level 4 log(SPB) 3 Iron Lyngby Agar, NMKL 96 2 FAST 1 0 day 1 day 5 day 11 Skjerdal og Ranneklev, 2006

25. Sushi study part 2:Does the preparation of sushi increase the risk that L. monocytogenes survival in stomach acid condition? Sushi prepared from • Acetic acid marinated rice (low pH) • Wasabi (also acidic) • Slices of raw fish • Main human barrier for food borne Listeria: Stomach acid • Reported in literature: Light acid stress leads to an increased tolerance to stronger acid.

26. Survival of L. monocytogenes in synthetic stomach acid Naturally contaminated salmon (<10 cfu/g) Sushi Undercooked minced fish raw fillet 10000 100 Listeria monocytogenes (cfu/g) 1 day 0 day 0 day 0 day 0 day 0 +1 day at 5°C +7 days at 0 +7 days at 0 7 days at 7°C + 1 day at 5°C +7 days at 7°C +7 days at 7°C storage of product prior to stomach acid condition exposure (time and temperature prior to acid exposure 10 min in acid 30 min in acid Acid adaptation and/or components in other sushi ingredients increase the tolerance of L. monocytogenes to stomach acid conditions stress Storage of fish for some days at abuse temperature (orange background) increase the risk of Listeria survival

27. Inoculated salmon Sushi Undercooked minced fish raw fillet 1000000 10000 Listeria monocytogenes (cfu/g) 100 1 day 0 day 0 day 0 day 0 day 0 5°C 7°C 7 days at +1 day at 5°C + 1 day at 7°C 7°C +7 days at +7 days at 0 +7 days at +7 days at 0 Storage of product prior to stomach acid condition exposure (time and temperature) prior to acid exposure 10 min in acid 30 min in acid Survival of L. monocytogenes in stomach acid conditions

28. Aspects to consider in food sampling (1):Contamination in the slaughter house and process environment • Origin categories: • Raw material (fish and water) • Human handling • Equipment, floor, roof etc (indirectly from humans or raw material) • Will the entire batch or only some fishes be contaminated? • Direct human contamination: Few fish • contaminated raw material and equipment: The entire batch may be contaminated The product from primary processing is the raw material in secondary processing

29. Aspects to consider in food sampling (2):Effects of processing, including preservation, on human pathogenic microbes in food • Preserved products are likely to contain higher levels of human pathogenic bacteria due to • Longer storage period – often at abuse temperature • Temporary process hurdles like light heat treatments remove spoilage flora but may also give the surviving bacteria a ”growth kick” if slowly chilled. • More processing leads to increased risk of contamination from humans • Product often consumed without traditional heat treatment or other treatments eliminating the microbes – ready-to-eat trend • etc

30. Consequenses of international trade and processing in seafood analysis and legislation • The food legislation including limit values for pathogens vary between countries (e. g. Listeria, zero tolerance in some countries, 100 cfu/g in others). • Some times there are different demands for semifinished products and final products. • The demands from the customers are often higher than those from the food authorities. • The same demands may be requested for seafood from different parts of the world, even though the pathogens of relevance differ in different parts of the world. • Limited traceability: suppliers and customers do not always trust each other. Indicator organisms for assessment of process hygiene, freshness and preservation, including masking, are important • Due to rapid spoilage and long travel distance: The analysis time and/or paper documents important for decisions.

31. Listeria risk management by food authorities • Give food advices to (vulnerable) consumers. • Upcoming: In Norway: Catch/slaughter day on label from 2010 • Upcoming: International legislation: Introduce performance objectives (PO) and food safety objectives (FSO) for intermediate products and final products, respectively. • What should the levels be? 10 cfu/g as PO for salmon intended used for ready-to-eat products and 100 cfu/g as FSO in final product? No Listeria in production environment samples if the fish that is going to be served to vulnerable consumers? • Relevant for other bacteria than Listeria • Many questions – some will be studied in the new EU project BASELINE starting in 2009.

32. Listeria risk management by companies • Keep Listeria levels as low as possible, both in products and production environment. • NB: Cleaning and desinfection may not be sufficient to control the level if there is a reservoar of L. monocytogenes in the processing plant. • Upcoming: International regulation (Europe at least): Companies will have to carry out risk assessment of products based on durability studies and/or literature data to prove that L. monocytogenes • Is not able to grow in the product, or • Can not reach maximum 100 cfu/g during the shelf life • More companies can do the studies together. Samples with naturally contaminated are most valuable. Studies started at VI already.

33. Summary • Seafood is healty food, and the solution to the food safety challenges is not to stop development of ready-to-eat products or international trade, but to manage the food safety risk risk. • Selection of good indicator parameters and analysis for their quantification are very important. • Thank you for your attention and invitation