São Paulo 2006

1. São Paulo 2006 Controlling Listeria monocytogenes in food Joaquín Martínez Suárez Departamento de Tecnología de Alimentos INIA Madrid Sao Paulo 2006

2. INIAInstituto Nacional de Investigación y Tecnología Agraria y Alimentaria National Institute for Agriculture and Food Research and Technology

3. INIA INIA is a public research organization belonging to the Spanish Ministry for Education and Science. INIA research and development activities focus on agriculture, food, forestry and environmental matters

4. DEPARTMENT OF FOOD TECHNOLOGY Research activities on different aspects related to the quality of dairy, meat and vegetable food products as well as on microbiological food safety Personnel: 35 Funding: 310.000 € • Research lines • Organoleptic quality and directed maturation of Spanish cheese through enzymatic and microbiological processes. • Processing and nutritional quality in Vegetable products. • Composition and quality in lamb meat. • Food microbiological safety using biological systems. Sao Paulo 2006

5. Department of Food Technology RESEARCH ON Listeria monocytogenes (Lm) • Detection • Characterization of field isolates from poultry and pork products and processing plants • Virulence testing of these isolates Sao Paulo 2006

6. Controlling Listeria monocytogenes in food Codex Alimentarius, 1996 ‘Although Listeria monocytogenes presence in many raw products cannot be prevented, because of its natural widespread occurrence in the environment, effective hygienic measures can reduce both the frequency and level of contamination’ Sao Paulo 2006

7. Controlling Listeria monocytogenes in food • Introduction to the organism • Specific detection of Listeria monocytogenes in foods • Monitoring for Listeria monocytogenes and tracing of the organism in food plants • Growthand survival of Listeria monocytogenes and microbiological challenge studies Sao Paulo 2006

8. Controlling Listeria monocytogenes in foodIntroduction to the organism • Taxonomy • Ecology • Virulence • Listeriosis • Outbreaks Sao Paulo 2006

9. Introduction to the organismTaxonomyMain laboratory tests for the differentiation of Listeria monocytogenes

10. Introduction to the organismTaxonomyGrouping of Listeria monocytogenes serotypes

11. water soil sewage decaying vegetation silage plants (cultivated and uncultivated) faeces (wild and domestic animals) humans mammals birds fish shellfish Introduction to the organismEcologySources of Listeria species Sao Paulo 2006

12. FACTORS host immunity number of cells ingested virulence of the specific strain HOST CELL INFECTION invasion multiplication with the host’s cell cytoplasm polymerization of host cell actin movement across the host’s cell cytoplasm invasion of adjacentcells Introduction to the organismVirulence Sao Paulo 2006

13. Introduction to the organismVirulence Are all the food isolates equally virulent? • mutations in virulence genes (inlA, hly, plcA, plcB, actA, prfA) • cell cultures • population genetics

14. Introduction to the organismListeriosis Invasive infection caused when contaminated food is ingested by susceptible individuals (infants, the elderly, pregnant, immuno-compromised) Symptoms vary, ranging from flu-like symptoms to meningitis and encephalitis Mortality rate for Listeriosis is 23% The infection usually occurs sporadically Sao Paulo 2006

15. Introduction to the organismOutbreaks

16. Risk factors Raw ingredients not subjected to a listericidal process or product susceptible to post-process contamination Refrigerated storage Product allowing growth of Listeria monocytogenes Extended shelf life (>10 days) Food is ready-to-eat Consumption by vulnerable groups Food Amount and frequency of consumption Frequency and level of contamination Introduction to the organismOutbreaks Sao Paulo 2006

17. Specific detection of Listeria monocytogenes in foods Sao Paulo 2006

18. Some commercial methods used for the specific detection or identification Listeria monocytogenes

19. Specific detection of Listeria monocytogenes in foodsChromogenic media Evaluation of ALOA plating medium for its suitabilityto recover high pressure-injured Listeria monocytogenesfrom ground chicken meat M.M. Jantzen1,2, J. Navas1, M. de Paz1, B. Rodríguez1, W.P. da Silva2, M. Nuñez1and J.V. Martínez-Suárez1 1 Departamento de Tecnología de Alimentos, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Madrid, Spain 2 Departamento de Ciência e Tecnologia Agroindustrial, Faculdade de Agronomia Eliseu Maciel, Universidade Federal de Pelotas, Pelotas, RS,Brazil Sao Paulo 2006

20. Specific detection of Listeria monocytogenes in foodsChromogenic media Evaluation of ALOA plating medium for its suitabilityto recover high pressure-injured Listeria monocytogenesfrom ground chicken meat

21. Alternative rapid methods for screening of enriched samples and/or culture confirmation Specific detection of Listeria monocytogenes in foods Sao Paulo 2006

22. Some commercial methods used for the specific detection or identification Listeria monocytogenes

23. Specific detection of Listeria monocytogenes in foods Immunoassay methodsELFA (Enzyme Linked Fluorescent Assay) Sao Paulo 2006

24. Specific detection of Listeria monocytogenes in foods Nucleic acid-based methodsDNA hybridization Sao Paulo 2006

25. Some commercial methods used for the specific detection or identification Listeria monocytogenes

26. Specific detection of Listeria monocytogenes in foods Nucleic acid-based methodsPCR Sao Paulo 2006

27. Specific detection of Listeria monocytogenes in foods Nucleic acid-based methodsPCR Influence of Enrichment Conditions on Real-Time PCR Detection of Listeria monocytogenes from Naturally Contaminated Ground Chicken Meat JAIME NAVAS, SAGRARIO ORTIZ, PILAR LOPEZ, MARCIA M. JANTZEN, VICTORIA LOPEZ, and JOAQUIN V. MARTINEZ-SUAREZ Sao Paulo 2006

28. Specific detection of Listeria monocytogenes in foods Nucleic acid-based methodsPCRInfluence of Enrichment Conditions on Real-Time PCR Detection of Listeria monocytogenes from Naturally Contaminated Ground Chicken Meat

29. Specific detection of Listeria monocytogenes in foods Future trends of Listeria monocytogenes detection methods Needs • Pre-detection methods to specifically concentrate Listeria monocytogenes cells • Tests targeting RNA Sao Paulo 2006

30. Specific detection of Listeria monocytogenes in foods Future trends of Listeria monocytogenes detection methods Novel methods • Microarrays or biochips • Biosensors Sao Paulo 2006

31. Specific detection of Listeria monocytogenes in foods CONCLUSIONSComparison of commercial methods for food testing for Listeria monocytogenesAdapted from Gasanov et al., 2005.

32. Specific detection of Listeria monocytogenes in foods CONCLUSIONS • Rapid methods means <4-5 days • All methods need a preenrichment step • PCR methods can detect L. monocytogenes in 28-30 hours • The selection of a technique depends on: • Type of sample • Sell by date of product (positive release system) Sao Paulo 2006

33. Monitoring for Listeria monocytogenes in food plantsand tracing of the organism FOOD MICROBIOLOGY • To prevent introduction of L. monocytogenes into food products • To prevent its multiplication • To identify its presence Sao Paulo 2006

34. Monitoring for Listeria monocytogenes in food plantsand tracing of the organism FOOD SAMPLES • incoming raw materials • food materials in process • finished products ENVIRONMENTAL SAMPLING TRACING THE ORGANISM Sao Paulo 2006

35. Monitoring for Listeria monocytogenes in food plantsand tracing of the organism Lm is widely distributed Raw material, destruction and recontamination Control of Lm • Sanitation • Kill organism in the plant environment • Both food contact and non-contact areas • Microbial testing • Validates that sanitation programs are effective in controlling Lm Sao Paulo 2006

36. FOOD SAMPLES incoming raw materials Harbor Lm raw, commercially available ground meat broiler carcasses turkey carcasses cow and bull carcasses hog carcasses Monitoring for Listeria monocytogenes in food plants and tracing of the organism Sao Paulo 2006

37. Monitoring for Listeria monocytogenes in food plants and tracing of the organism FOOD SAMPLES A small percentage of certain foods are contaminated generally at very low levels Sao Paulo 2006

38. Monitoring for Listeria monocytogenes in food plants and tracing of the organismFOOD SAMPLESA small percentage of certain foods are contaminated generally at very low levels L. monocytogenes in ready-to-eat foods at retail (Gombas et al., 2003)

39. Monitoring for Listeria monocytogenes in food plantsand tracing of the organism FOOD SAMPLES • IN-PROCESS PRODUCT TESTING Sao Paulo 2006

40. Monitoring for Listeria monocytogenes in food plantsand tracing of the organism FOOD SAMPLES END-PRODUCT TESTING Sao Paulo 2006

41. Monitoring for Listeria monocytogenes in food plantsand tracing of the organism ENVIRONMENTAL SAMPLING Food processors should minimize contamination • GMPs • SSOPs • HACCP • Separation of cooked from raw foods Sao Paulo 2006

42. Monitoring for Listeria monocytogenes in food plantsand tracing of the organism ENVIRONMENTAL SAMPLING • sampling to minimize environmental contamination with Lm • effective plan to take corrective action Sao Paulo 2006

43. Monitoring for Listeria monocytogenes in food plantsand tracing of the organism ENVIRONMENTAL SAMPLING L. monocytogenes or Listeria spp. • Test for Listeria species as an indicator of the potential presence of Lm • More likely to find • Lm not frequently found and not uniformly distributed Sao Paulo 2006

44. ENVIRONMENTAL SAMPLING NON-FOOD CONTACT SURFACES Common reservoirs Floors Drains Walls Ceilings Overhead structures Catwalks Insulation Cleaning tools Maintenance tools Equipment framework Condensate Monitoring for Listeria monocytogenes in food plantsand tracing of the organism Sao Paulo 2006

45. ENVIRONMENTAL SAMPLING FOOD CONTACT SURFACES Filling/packaging equipment Conveyors, belts Solutions used to chill food Slicers, dicers, shredders, blenders Collators used for assembling/arranging produce Shelves Racks used for transport Spiral/blast freezers Food containers used for transport Monitoring for Listeria monocytogenes in food plantsand tracing of the organism Sao Paulo 2006

46. Monitoring for Listeria monocytogenes in food plantsand tracing of the organism Listeria monocytogenes subtyping methods • PCR-serotyping • Pulsed field gel electrophoresis (PFGE) • Ribotyping • Multilocus sequence typing (MLST) Sao Paulo 2006

47. Monitoring for Listeria monocytogenes in food plantsand tracing of the organism Listeria monocytogenes subtyping methods • PCR-serotyping 1/2 a, 1/2b, 1/2c, and 4b Sao Paulo 2006

48. Monitoring for Listeria monocytogenes in food plantsand tracing of the organism Listeria monocytogenes subtyping methods PCR-serotyping (Doumith et al., 2004): targets and products

49. Monitoring for Listeria monocytogenes in food plantsand tracing of the organism Listeria monocytogenes subtyping methods PCR-serotyping (Doumith et al., 2004) • 1-12 L. monocytogenes • 1/2a • 1/2b • 3 1/2c • 4 4b

50. Monitoring for Listeria monocytogenes in food plantsand tracing of the organism Listeria monocytogenes subtyping methods Pulsed field gel electrophoresis (PFGE) Sao Paulo 2006