Sao Paulo 2006Sao Paulo 2006 11
São PauloSão Paulo 2006 2006
Controlling Controlling
Listeria monocytogenesListeria monocytogenes in in foodfood
Joaquín Martínez SuárezJoaquín Martínez Suárez
Departamento de Tecnología de AlimentosDepartamento de Tecnología de Alimentos
INIAINIA
MadridMadrid
INIAInstituto Nacional de Investigación y
Tecnología Agraria y Alimentaria
National Institute for Agriculture and Food Research and Technology
INIAINIAINIA is a public research INIA is a public research organization belonging to organization belonging to the Spanish Ministry for the Spanish Ministry for Education and Science.Education and Science.
INIA research and INIA research and development activities development activities focus on agriculture, focus on agriculture, food, forestry and food, forestry and environmental mattersenvironmental matters
Sao Paulo 2006Sao Paulo 2006 44
DEPARTMENT OF FOOD TECHNOLOGYDEPARTMENT OF FOOD TECHNOLOGY
Research activities on different aspects related to Research activities on different aspects related to the the quality of quality of dairy, meat and vegetable dairy, meat and vegetable food food products as well as on products as well as on microbiological food safetymicrobiological food safety
Personnel: 35Personnel: 35 Funding: 310.000 €Funding: 310.000 €
Research linesResearch 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 2006Sao Paulo 2006 55
Department of Food TechnologyDepartment of Food Technology
RESEARCH ON RESEARCH ON Listeria monocytogenes (Lm)Listeria monocytogenes (Lm)
DetectionDetection
Characterization of field isolates from poultry and pork Characterization of field isolates from poultry and pork products and processing plantsproducts and processing plants
Virulence testing of these isolatesVirulence testing of these isolates
Sao Paulo 2006Sao Paulo 2006 66
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in food
Codex Alimentarius, 1996Codex Alimentarius, 1996
‘‘Although Although Listeria monocytogenesListeria monocytogenes presence in many raw presence in many raw products cannot be prevented, because of its natural products cannot be prevented, because of its natural widespread occurrence in the environment, effective widespread occurrence in the environment, effective
hygienic measures can reduce both the frequency and hygienic measures can reduce both the frequency and level of contamination’level of contamination’
Sao Paulo 2006Sao Paulo 2006 77
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in food
Introduction to the organismIntroduction to the organism
Specific Specific detectiondetection of of Listeria monocytogenesListeria monocytogenes in foods in foods
Monitoring for Monitoring for ListeriaListeria monocytogenes and monocytogenes and tracingtracing of the of the organism in food plantsorganism in food plants
GrowthGrowth and survival of and survival of Listeria monocytogenes Listeria monocytogenes and and microbiological challenge studiesmicrobiological challenge studies
Sao Paulo 2006Sao Paulo 2006 88
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in food
Introduction to the organismIntroduction to the organism
TaxonomyTaxonomy EcologyEcology VirulenceVirulence ListeriosisListeriosis OutbreaksOutbreaks
Introduction to the organismTaxonomy
Main laboratory tests for the differentiation of Listeria monocytogenes
Species Haemolysis Phosphatidylinositol phospholipase C
Acid produced from
L-Rhamnose D-Xylose
L. monocytogenes + + + - L. ivanovii + + - + L. seeligeri + - - + L. innocua - - Variable - L. welshimeri - - Variable + L. grayi - - Variable -
Introduction to the organismIntroduction to the organismTaxonomyTaxonomy
Grouping of Grouping of Listeria monocytogenesListeria monocytogenes serotypes serotypes
Lineage 1 Lineage 2 Lineage 3
1/2a 1/2b 4a
1/2c 3b 4c
3a 4b
3c 4d
4e
Sao Paulo 2006Sao Paulo 2006 1111
Introduction to the organismIntroduction to the organismEcologyEcology
Sources of Sources of Listeria Listeria speciesspecies
waterwater soilsoil sewagesewage decaying decaying
vegetationvegetation silagesilage plants (cultivated plants (cultivated
and uncultivated)and uncultivated)
faeces (wild and domestic faeces (wild and domestic animals)animals)
humanshumans mammalsmammals birdsbirds fishfish shellfish shellfish
Sao Paulo 2006Sao Paulo 2006 1212
Introduction to the organismIntroduction to the organism
VirulenceVirulence
FACTORSFACTORS
host immunityhost immunity number of cells ingestednumber of cells ingested virulence of the specific virulence of the specific
strainstrain
HOST CELL INFECTIONHOST CELL INFECTION
invasioninvasion multiplication with the multiplication with the
host’s cell cytoplasmhost’s cell cytoplasm polymerization of host polymerization of host
cell actincell actin movement across the movement across the
host’s cell cytoplasmhost’s cell cytoplasm invasion of adjacentinvasion of adjacent
cellscells
Introduction to the organismIntroduction to the organismVirulenceVirulence
Are all the food Are all the food isolates isolates
equally virulent?equally virulent?
mutations in virulence mutations in virulence genes (genes (inlA, hly, plcA, inlA, hly, plcA, plcB, actA, prfA)plcB, actA, prfA)
cell culturescell cultures
population geneticspopulation genetics
Sao Paulo 2006Sao Paulo 2006 1414
Introduction to the organismIntroduction to the organismListeriosisListeriosis
Invasive infection caused when contaminated food is Invasive infection caused when contaminated food is ingested by susceptible individuals (infants, the elderly, ingested by susceptible individuals (infants, the elderly, pregnant, immuno-compromised)pregnant, immuno-compromised)
Symptoms vary, ranging from flu-like symptoms to Symptoms vary, ranging from flu-like symptoms to meningitis and encephalitismeningitis and encephalitis
Mortality rate for Listeriosis is 23%Mortality rate for Listeriosis is 23%
The infection usually occurs sporadicallyThe infection usually occurs sporadically
Introduction to the organismIntroduction to the organismOutbreaksOutbreaks
Year Country Cases (deaths)
Food Serotype Reference
1980-81 Canada 41 (18)
Coleslaw 4b Schlech et al. (1983)
1983-87 Switzerland 122 (34)
Vacherin cheese 4b Bille (1990)
1987-89 UK >350 (>90)
Belgian paté 4b McLauchlin et al. (1991)
1992 New Zealand 4 (2)
Smoked mussels 1/2a & 4 Baker et al. (1993)
1992 France 279 (63)
Pork tongue in aspic 4b Goulet et al. (1993)
1994 USA 45 (0)
Chocolate milk 1/2b Dalton et al. (1997)
1997 Italy 1566 (0)
Corn and tuna salad 4b Aureli et al. (2000)
1998-99 Finland 25 (6)
Butter 3a Lyytikäinen et al. (2000)
Sao Paulo 2006Sao Paulo 2006 1616
Introduction to the organismIntroduction to the organismOutbreaksOutbreaks
Risk factorsRisk factors
Raw ingredients not subjected Raw ingredients not subjected to a listericidal process or to a listericidal process or product susceptible to post-product susceptible to post-process contaminationprocess contamination
Refrigerated storageRefrigerated storage Product allowing growth of Product allowing growth of
Listeria monocytogenesListeria monocytogenes Extended shelf life (>10 days)Extended shelf life (>10 days) Food is ready-to-eatFood is ready-to-eat Consumption by vulnerable Consumption by vulnerable
groupsgroups
FoodFood
Amount and frequency Amount and frequency of consumptionof consumption
Frequency and level of Frequency and level of contaminationcontamination
Sao Paulo 2006Sao Paulo 2006 1717
Specific detection of Specific detection of Listeria monocytogenesListeria monocytogenes in foods in foods
Some commercial methods used for the specific detection or identification Some commercial methods used for the specific detection or identification Listeria monocytogenesListeria monocytogenes
Type of method Analytical Technique Test name
Culture Chromogenic medium ALOA (also known under other names)
Chromogenic medium Rapid L.Mono®
Immunoassay ELFA VIDAS® LMO
ELISA Transia® Plate Listeria monocytogenes
Molecular method Nucleic acid hybridization probe
Gene Trak® and GeneQuench® Listeria monocytogenes Assays
Nucleic acid hybridization probe
AccuProbe® Listeria monocytogenes
Real-time PCR BAX® L. monocytogenes Detection System
Real-time PCR Probelia® Listeria monocytogenes PCR System
Real-time PCR LightCycler® Listeria monocytogenes Detection kit
Real-time PCR TaqMan® Listeria monocytogenes Detection Kit
Real-time PCR GeneVision® Rapid Pathogen Detection System for Listeria monocytogenes
Sao Paulo 2006Sao Paulo 2006 1919
Specific detection of Specific detection of Listeria monocytogenesListeria monocytogenes in foods in foods
Chromogenic media Chromogenic media
Evaluation of ALOA plating medium for its suitabilityto recover high pressure-injured Listeria monocytogenes
from ground chicken meat
M.M. Jantzen1,2, J. Navas1, M. de Paz1, B. Rodríguez1, W.P. da Silva2, M. Nuñez1
and 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
Specific detection of Specific detection of Listeria monocytogenesListeria monocytogenes in foods in foods
Chromogenic media Chromogenic media
Evaluation of ALOA plating medium for its suitabilityto recover high pressure-injured Listeria monocytogenes
from ground chicken meat
Sao Paulo 2006Sao Paulo 2006 2121
Specific detection of Specific detection of Listeria monocytogenesListeria monocytogenes in foods in foods
Alternative rapid methods for Alternative rapid methods for screening of enriched samples screening of enriched samples
and/or culture confirmationand/or culture confirmation
Some commercial methods used for the specific detection or identification Some commercial methods used for the specific detection or identification Listeria monocytogenesListeria monocytogenes
Type of method Analytical Technique Test name
Culture Chromogenic medium ALOA (also known under other names)
Chromogenic medium Rapid L.Mono®
Immunoassay ELFA VIDAS® LMO
ELISA Transia® Plate Listeria monocytogenes
Molecular method Nucleic acid hybridization probe
Gene Trak® and GeneQuench® Listeria monocytogenes Assays
Nucleic acid hybridization probe
AccuProbe® Listeria monocytogenes
Real-time PCR BAX® L. monocytogenes Detection System
Real-time PCR Probelia® Listeria monocytogenes PCR System
Real-time PCR LightCycler® Listeria monocytogenes Detection kit
Real-time PCR TaqMan® Listeria monocytogenes Detection Kit
Real-time PCR GeneVision® Rapid Pathogen Detection System for Listeria monocytogenes
Sao Paulo 2006Sao Paulo 2006 2323
Specific detection of Specific detection of Listeria monocytogenesListeria monocytogenes in foods in foods Immunoassay methodsImmunoassay methods
ELFA (Enzyme Linked Fluorescent Assay)ELFA (Enzyme Linked Fluorescent Assay)
Sao Paulo 2006Sao Paulo 2006 2424
Specific detection of Specific detection of Listeria monocytogenesListeria monocytogenes in foods in foods Nucleic acid-based methodsNucleic acid-based methods
DNA hybridizationDNA hybridization
Some commercial methods used for the specific detection or identification Some commercial methods used for the specific detection or identification Listeria monocytogenesListeria monocytogenes
Type of method Analytical Technique Test name
Culture Chromogenic medium ALOA (also known under other names)
Chromogenic medium Rapid L.Mono®
Immunoassay ELFA VIDAS® LMO
ELISA Transia® Plate Listeria monocytogenes
Molecular method Nucleic acid hybridization probe
Gene Trak® and GeneQuench® Listeria monocytogenes Assays
Nucleic acid hybridization probe
AccuProbe® Listeria monocytogenes
Real-time PCR BAX® L. monocytogenes Detection System
Real-time PCR Probelia® Listeria monocytogenes PCR System
Real-time PCR LightCycler® Listeria monocytogenes Detection kit
Real-time PCR TaqMan® Listeria monocytogenes Detection Kit
Real-time PCR GeneVision® Rapid Pathogen Detection System for Listeria monocytogenes
Sao Paulo 2006Sao Paulo 2006 2626
Specific detection of Specific detection of Listeria monocytogenesListeria monocytogenes in foods in foods Nucleic acid-based methodsNucleic acid-based methods
PCRPCR
Sao Paulo 2006Sao Paulo 2006 2727
Specific detection of Specific detection of Listeria monocytogenesListeria monocytogenes in foods in foods Nucleic acid-based methodsNucleic acid-based methods
PCRPCR
Influence of Enrichment Conditions on Real-Influence of Enrichment Conditions on Real-Time PCR Detection of Time PCR Detection of Listeria Listeria monocytogenesmonocytogenes from Naturally from Naturally
Contaminated Ground Chicken MeatContaminated Ground Chicken Meat
JAIME NAVAS, SAGRARIO ORTIZ, PILAR LOPEZ, MARCIA M. JAIME NAVAS, SAGRARIO ORTIZ, PILAR LOPEZ, MARCIA M. JANTZEN, VICTORIA LOPEZ, and JOAQUIN V. MARTINEZ-JANTZEN, VICTORIA LOPEZ, and JOAQUIN V. MARTINEZ-
SUAREZSUAREZ
Specific detection of Specific detection of Listeria monocytogenesListeria monocytogenes in foods in foods Nucleic acid-based methodsNucleic acid-based methods
PCRPCRInfluence of Enrichment Conditions on Real-Time PCR Detection of Influence of Enrichment Conditions on Real-Time PCR Detection of
Listeria monocytogenesListeria monocytogenes from Naturally Contaminated Ground Chicken from Naturally Contaminated Ground Chicken MeatMeat
Enrichment type
Conditions
No. of
samples tested
No. (%)
positive on subculture to ALOA
No. (%) positive in real-
time PCR
No. (%) positive by either method
No. (%) positive by both methods
No. (%)
concordant
Primary UVM1, 24 h, 30ºC
37 26 (70.2) 14 (37.8) 28 (75.6) 11 (29.7) 20 (54.0)
Secondary MOPS-BLEB, 24 h, 37ºC
37 20 (54.0) 26 (70.2) 26 (70.2) 20 (54.0) 31 (83.7)
USDA-FSIS Primary plus secondary for culture and
secondary for PCR
37 26 (70.2) 26 (70.2) 27 (72.9) 26 (70.2) 35 (94.5)
Sao Paulo 2006Sao Paulo 2006 2929
Specific detection of Listeria monocytogenes in foods Specific detection of Listeria monocytogenes in foods
Future trends of Listeria monocytogenes detection methodsFuture trends of Listeria monocytogenes detection methods
NeedsNeeds
Pre-detection methods to specifically Pre-detection methods to specifically concentrate concentrate Listeria monocytogenesListeria monocytogenes cells cells
Tests targeting RNATests targeting RNA
Sao Paulo 2006Sao Paulo 2006 3030
Specific detection of Listeria monocytogenes in foods Specific detection of Listeria monocytogenes in foods
Future trends of Listeria monocytogenes detection methodsFuture trends of Listeria monocytogenes detection methods
Novel methodsNovel methods
Microarrays or Microarrays or biochipsbiochips
BiosensorsBiosensors
Specific detection of Specific detection of Listeria monocytogenesListeria monocytogenes in foods in foods
CONCLUSIONSCONCLUSIONS
Comparison of commercial methods for food testing for Comparison of commercial methods for food testing for Listeria monocytogenesListeria monocytogenes
Adapted from Gasanov Adapted from Gasanov et alet al., 2005. ., 2005.
Type of method
Analytical Technique Approximate time (h)
Main use and primary matrices
Sensitivity
(cells/mL)
Culture Chromogenic medium 24-48 Isolation, food ≤10 4
Immunoassay ELFA 1-2 Screening, food 105
Molecular method
Nucleic acid hybridization probe
2-4 Screening, food and environmental
samples
107
Real-time PCR 2 Screening, food 105
Sao Paulo 2006Sao Paulo 2006 3232
Specific detection of Specific detection of Listeria monocytogenesListeria monocytogenes in foods in foods
CONCLUSIONSCONCLUSIONS
1.1. Rapid methods means <4-5 daysRapid methods means <4-5 days
2.2. All methods need a preenrichment stepAll methods need a preenrichment step
3.3. PCR methods can detect PCR methods can detect L. monocytogenesL. monocytogenes in in 28-30 hours28-30 hours
4.4. The selection of a technique depends on:The selection of a technique depends on:
Type of sampleType of sample
Sell by date of product (positive release Sell by date of product (positive release system)system)
Sao Paulo 2006Sao Paulo 2006 3333
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plants and tracing of the organismand tracing of the organism
FOOD MICROBIOLOGYFOOD MICROBIOLOGY
To prevent introduction of To prevent introduction of L. monocytogenesL. monocytogenes into into food productsfood products
To prevent its To prevent its multiplicationmultiplication
To identify its presenceTo identify its presence
Sao Paulo 2006Sao Paulo 2006 3434
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plants and tracing of the organismand tracing of the organism
FOOD SAMPLESFOOD SAMPLES
incoming raw materialsincoming raw materials food materials in processfood materials in process finished productsfinished products
ENVIRONMENTAL ENVIRONMENTAL SAMPLINGSAMPLING
TRACING THE ORGANISMTRACING THE ORGANISM
Sao Paulo 2006Sao Paulo 2006 3535
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plants and tracing of the organismand tracing of the organism
Lm is widely distributedLm is widely distributed
Raw material, destruction and Raw material, destruction and recontaminationrecontamination
Control of LmControl of Lm SanitationSanitation
• Kill organism in the plant environmentKill organism in the plant environment Both food contact and non-contact areasBoth food contact and non-contact areas
Microbial testingMicrobial testing• Validates that sanitation programs are effective in Validates that sanitation programs are effective in
controlling Lmcontrolling Lm
Sao Paulo 2006Sao Paulo 2006 3636
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food in food plants and tracing of the organismplants and tracing of the organism
FOOD SAMPLESFOOD SAMPLES incoming raw incoming raw
materialsmaterials
Harbor Harbor LmLm raw, commercially raw, commercially
available ground available ground meatmeat
broiler carcassesbroiler carcasses turkey carcassesturkey carcasses cow and bull cow and bull
carcassescarcasses hog carcasseshog carcasses
Sao Paulo 2006Sao Paulo 2006 3737
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food in food plants and tracing of the organismplants and tracing of the organism
FOOD SAMPLESFOOD SAMPLES
A small percentage of certain A small percentage of certain foods are contaminated foods are contaminated generally at very low levels generally at very low levels
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plants and tracing of the organismand tracing of the organism
FOOD SAMPLESFOOD SAMPLESA small percentage of certain foods are contaminated generally at very low levels A small percentage of certain foods are contaminated generally at very low levels
L. monocytogenesL. monocytogenes in ready-to-eat foods at retail (Gombas in ready-to-eat foods at retail (Gombas et alet al., 2003)., 2003)
Product category % positive
Deli salads 2.36
Deli meats 0.89
Fresh soft cheeses 0.17
Bagged salads 0.74
Blue-veined cheeses 1.41
Mold-ripened cheeses 1.03
Seafood salads 4.70
Smoked seafood 4.31
TOTAL 1.81
Sao Paulo 2006Sao Paulo 2006 3939
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organismand tracing of the organism
FOOD SAMPLESFOOD SAMPLES
IN-PROCESS IN-PROCESS PRODUCT TESTINGPRODUCT TESTING
Sao Paulo 2006Sao Paulo 2006 4040
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organismand tracing of the organism
FOOD SAMPLESFOOD SAMPLES
END-PRODUCT END-PRODUCT TESTINGTESTING
Sao Paulo 2006Sao Paulo 2006 4141
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organismand tracing of the organism
ENVIRONMENTAL SAMPLINGENVIRONMENTAL SAMPLING
Food processors should minimize contaminationFood processors should minimize contamination
• GMPsGMPs• SSOPsSSOPs• HACCPHACCP• Separation of cooked from raw foodsSeparation of cooked from raw foods
Sao Paulo 2006Sao Paulo 2006 4242
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organismand tracing of the organism
ENVIRONMENTAL ENVIRONMENTAL SAMPLINGSAMPLING
sampling to minimize sampling to minimize environmental environmental contamination with Lmcontamination with Lm
effective plan to take effective plan to take corrective actioncorrective action
Sao Paulo 2006Sao Paulo 2006 4343
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organismand tracing of the organism
ENVIRONMENTAL SAMPLINGENVIRONMENTAL SAMPLING
L. monocytogenesL. monocytogenes or or ListeriaListeria spp. spp.
Test for Test for ListeriaListeria species as an species as an indicator of the potential indicator of the potential presence of Lmpresence of Lm
More likely to findMore likely to find
Lm not frequently found and not Lm not frequently found and not uniformly distributed uniformly distributed
Sao Paulo 2006Sao Paulo 2006 4444
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organismand tracing of the organism
ENVIRONMENTAL ENVIRONMENTAL SAMPLINGSAMPLING
NON-FOOD CONTACT SURFACESNON-FOOD CONTACT SURFACES
Common reservoirsCommon reservoirs FloorsFloors DrainsDrains WallsWalls CeilingsCeilings Overhead structuresOverhead structures CatwalksCatwalks InsulationInsulation Cleaning toolsCleaning tools Maintenance toolsMaintenance tools Equipment frameworkEquipment framework CondensateCondensate
Sao Paulo 2006Sao Paulo 2006 4545
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organismand tracing of the organism
ENVIRONMENTAL ENVIRONMENTAL SAMPLINGSAMPLING
FOOD CONTACT SURFACESFOOD CONTACT SURFACES
Filling/packaging Filling/packaging equipmentequipment
Conveyors, beltsConveyors, belts Solutions used to chill foodSolutions used to chill food Slicers, dicers, shredders, Slicers, dicers, shredders,
blendersblenders Collators used for Collators used for
assembling/arranging assembling/arranging produceproduce
ShelvesShelves Racks used for transportRacks used for transport Spiral/blast freezersSpiral/blast freezers Food containers used for Food containers used for
transporttransport
Sao Paulo 2006Sao Paulo 2006 4646
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organismand tracing of the organism
Listeria monocytogenesListeria monocytogenes subtyping methodssubtyping methods
PCR-serotypingPCR-serotyping Pulsed field gel electrophoresis Pulsed field gel electrophoresis
(PFGE)(PFGE) RibotypingRibotyping
Multilocus sequence typing (MLST)Multilocus sequence typing (MLST)
Sao Paulo 2006Sao Paulo 2006 4747
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organismand tracing of the organism
Listeria monocytogenesListeria monocytogenes
subtyping methodssubtyping methods
PCR-serotypingPCR-serotyping
1/2 a, 1/2b, 1/2c, and 4b1/2 a, 1/2b, 1/2c, and 4b
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organismand tracing of the organism
Listeria monocytogenesListeria monocytogenes subtyping methods subtyping methods
PCR-serotyping (Doumith PCR-serotyping (Doumith et alet al., 2004): ., 2004): targets and productstargets and products
Gene (protein) PCR product
(bp)
Serovar specificity
lmo0737 (unknown)
691 L. monocytogenes 1/2a, 1/2c, 3a, 3c
lmo1118 (unknown)
906 L. monocytogenes 1/2c, 3c
ORF2819 (putative transcriptional regulator)
471 L. monocytogenes 1/2b, 3b, 4b, 4d, 4e
ORF2110 (putative secreted protein)
597 L. monocytogenes 4b, 4d, 4e
prs (putative PRPP synthetase)
370 Listeria spp.
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organismand tracing of the organism
Listeria monocytogenesListeria monocytogenes subtyping methods subtyping methods
PCR-serotyping (Doumith PCR-serotyping (Doumith et alet al., 2004)., 2004)
1-12 L. monocytogenes
1 1/2a 2 1/2b 3 1/2c 4 4b
Sao Paulo 2006Sao Paulo 2006 5050
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organismand tracing of the organism
Listeria monocytogenesListeria monocytogenes subtyping methodssubtyping methods
Pulsed field gel Pulsed field gel electrophoresis (PFGE)electrophoresis (PFGE)
Sao Paulo 2006Sao Paulo 2006 5151
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organismand tracing of the organism
Listeria monocytogenesListeria monocytogenes subtyping methods subtyping methods
RibotypingRibotyping
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organismand tracing of the organism
Listeria monocytogenesListeria monocytogenes subtyping methods subtyping methods
Multilocus sequence typing (MLST)Multilocus sequence typing (MLST)
Sao Paulo 2006Sao Paulo 2006 5353
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organismand tracing of the organism
3 studies on poultry 3 studies on poultry contamination:contamination:
AbattoirAbattoir Processing plantProcessing plant RetailRetail
Sao Paulo 2006Sao Paulo 2006 5454
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organismand tracing of the organism
Molecular subtyping of Molecular subtyping of
Listeria monocytogenesListeria monocytogenes originating from a originating from a broiler abattoir broiler abattoir
Victoria López, Sagrario Ortiz, Alfredo Corujo, Pilar López, Victoria López, Sagrario Ortiz, Alfredo Corujo, Pilar López, Jaime Navas, Raúl Moreno, Joaquín V. Martínez-SuárezJaime Navas, Raúl Moreno, Joaquín V. Martínez-Suárez
Sao Paulo 2006Sao Paulo 2006 5555
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organismand tracing of the organism
Molecular subtyping of Molecular subtyping of Listeria monocytogenesListeria monocytogenes originating from a originating from a broiler abattoir broiler abattoir
Sampling resultsSampling results
MonthMonth Nº LmNº Lm 1/2a1/2a 1/2b1/2b
MarchMarch 1010 2 2 (non-contact)(non-contact)
88
AprilApril 1919 11(non-contact)(non-contact)
1818
MayMay 2020 2 2 (non-contact)(non-contact)
1818
AscI ApaI
S D A B S C H E J S S 1 2 4 5 6 S 7 3 8 9 S
- 622 Kb
- 220 Kb
- 37 Kb
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organismand tracing of the organism
Molecular subtyping of Molecular subtyping of Listeria monocytogenesListeria monocytogenes originating from a originating from a broiler abattoir broiler abattoir
Pulsed field gel electrophoresis (PFGE)Pulsed field gel electrophoresis (PFGE)
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organismand tracing of the organism
Molecular subtyping of Molecular subtyping of Listeria monocytogenesListeria monocytogenes originating from a originating from a broiler abattoir broiler abattoir
Asc I / Apa I pulsotypes
0
1
2
3
4
5
6
7
8
Canales Contacto No contacto
0
1
2
3
4
5
6
7
8
Canales Contacto No contacto
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
P11
P12
0
1
2
3
4
5
6
7
8
Canales Contacto No contacto
Sao Paulo 2006Sao Paulo 2006 5858
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organismand tracing of the organism
Source of the contamination Source of the contamination with with Listeria monocytogenesListeria monocytogenes
serotype 4b in processed serotype 4b in processed chicken productschicken products
Victoria López, Sagrario Ortiz, Alfredo Corujo, Victoria López, Sagrario Ortiz, Alfredo Corujo, Pilar López, Jaime Navas, Raúl Moreno, and Pilar López, Jaime Navas, Raúl Moreno, and
Joaquín V. Martínez-SuárezJoaquín V. Martínez-Suárez
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organism and tracing of the organism
Source of the contamination with Source of the contamination with Listeria monocytogenesListeria monocytogenes serotype 4b in serotype 4b in processed chicken productsprocessed chicken products
Distribution of the isolates of Distribution of the isolates of L. monocytogenesL. monocytogenes found in different found in different retail broiler products, August of 2005retail broiler products, August of 2005
Serotype Nº Isolates Products Nº of pulsotypes
AscI/ ApaI
4b 14 chicken meatball, chicken brochette, frozen
chicken burguer, chicken burguer, red chicken
sausage and “flamenquin”
1
1/2c 4 chicken brochette and 100% chicken sausage 2
1/2b 5 ”Butifarra” and “carne de pincho” 3
1/2a 57 chicken meatball, chicken wings, chicken sausage,
chicken burguer, chicken burger 100%, red
chicken sausage, “mini flamenquin”, roast chicken
9
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organism and tracing of the organism
Source of the contamination with Source of the contamination with Listeria monocytogenesListeria monocytogenes serotype 4b in serotype 4b in processed chicken productsprocessed chicken products
Serotypes and PFGE profiles of theSerotypes and PFGE profiles of the Listeria monocytogenes Listeria monocytogenes isolates from pork fatisolates from pork fat
Serotype Nº isolates Pulsotype Products with this pulsotype
4b 8 A chicken meatball chicken brochette
frozen chicken burguer chicken burguer
red chicken sausage “flamenquin”
1/2c 12 B natural brochette sausage 100%
1/2b 4 C and D Not found
Sao Paulo 2006Sao Paulo 2006 6161
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organism and tracing of the organism
Navas, J., Ortiz, S., López, P., López, V., Martínez-Suárez, J.V.
Listeria monocytogenesListeria monocytogenes in poultry products in poultry products
at retailat retail
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organism and tracing of the organism
Listeria monocytogenesListeria monocytogenes in poultry products at retail in poultry products at retail
AscI profiles
ApaI profiles
AscI/ApaI PULSOTYPES SEROTYPE Nº SAMPLES
1 A 1A I 1/2c 12
1 H 1H II 1/2c 2
2 B 2B III 1/2b 2
3 C 3C IV 1/2a 3
4 B 4B V 1/2b 9
5 D 5D VI 1/2b 3
5 F 5F VII 1/2b 1
6 E 6E VIII 1/2a 2
7 C 7C IX 1/2a 2
8 I 8I X 1/2a 1
9 C 9C XI 1/2a 4
10 G 10G XII 1/2c 2
11 J 11J XIII 1/2b 2
Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organism and tracing of the organism
Listeria monocytogenesListeria monocytogenes in poultry products at retail in poultry products at retail
ApaApaI resultsI results
Sao Paulo 2006Sao Paulo 2006 6464
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in food
Growth and survivalGrowth and survival
Preventing growth of Lm in contaminated Preventing growth of Lm in contaminated ready-to-eat foods through time and ready-to-eat foods through time and
temperature controls, or through temperature controls, or through reformulation of foods so that they reformulation of foods so that they
retard or do not support growth of Lmretard or do not support growth of Lm
Sao Paulo 2006Sao Paulo 2006 6565
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in food
Growth and survivalGrowth and survival
Factors affecting the growth and survival of Lm
•temperature•pH•water activity
Products not capable of supporting the growth of Lm
Stress responses
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in food
Growth and survivalGrowth and survival
Limits for the growth of Limits for the growth of Listeria monocytogenesListeria monocytogenes (ICMSF, 1996) (ICMSF, 1996)
Parameter Minimum Maximum
Temperature (ºC) -0.4 45
pH 4.39 9.4
Water activity (aw) 0.92 -
Sao Paulo 2006Sao Paulo 2006 6767
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in foodGrowth and survivalGrowth and survival
Products not capable of supporting the Products not capable of supporting the growth of Lm growth of Lm
1. Water activity (Aw) value of less than 0.922. pH less than 4.39 when measured at 24ºC3. Food in an unopened, sealed container that is
commercially sterile under non-refrigerated storage4. Laboratory evidence demonstrates that no growth of
Lm can occur5. The product does not support the growth of any
microorganisms
Sao Paulo 2006Sao Paulo 2006 6868
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in foodGrowth and survival of Listeria monocytogenes Growth and survival of Listeria monocytogenes
Stress responsesStress responses
Heat-shock response and acid adaptationHeat-shock response and acid adaptation
Sao Paulo 2006Sao Paulo 2006 6969
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in food
Growth and survivalGrowth and survival
Combining factorsCombining factors
Other means for Other means for controlling Lmcontrolling Lm
Sao Paulo 2006Sao Paulo 2006 7070
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in food
Growth and survivalGrowth and survival
Other means for controlling LmOther means for controlling Lm
Currently used chemicalsCurrently used chemicals
Sodium hypochloriteSodium hypochlorite Gas atmosphereGas atmosphere Sodium nitriteSodium nitrite Other antimicrobial chemicals (sodium Other antimicrobial chemicals (sodium
lactate, sodium acetate, sodium diacetate, lactate, sodium acetate, sodium diacetate, sodium benzoate, sodium propionate, sodium benzoate, sodium propionate, potassium sorbate) potassium sorbate)
Sao Paulo 2006Sao Paulo 2006 7171
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in food
Growth and survivalGrowth and survival
Other means for controlling LmOther means for controlling Lm
Currently used nCurrently used nonthermal technologiesonthermal technologies
Nisin and other bacteriocinsNisin and other bacteriocins Gamma irradiationGamma irradiation High-hydrostatic-pressure technologyHigh-hydrostatic-pressure technology
Sao Paulo 2006Sao Paulo 2006 7272
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in food
Growth and survivalGrowth and survival
Other means for controlling LmOther means for controlling Lm
Novel technologies: Chemical productsNovel technologies: Chemical products
• Hydrogen peroxideHydrogen peroxide• Calcinated calciumCalcinated calcium• Plant essential oilsPlant essential oils• OzoneOzone• Acidic calcium sulfate, lactic acid and propionic acidAcidic calcium sulfate, lactic acid and propionic acid• Peracetic acid (15%) and H2O2 (10%)Peracetic acid (15%) and H2O2 (10%)• Peroxyacetic acid-based additive Peroxyacetic acid-based additive • Sodium or potassium lactate and sodium diacetateSodium or potassium lactate and sodium diacetate• Chlorine dioxideChlorine dioxide
Sao Paulo 2006Sao Paulo 2006 7373
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in food
Growth and survivalGrowth and survival
Other means for controlling LmOther means for controlling Lm
Novel technologies:Physical Novel technologies:Physical treatmentstreatments
Surface pasteurization technologySurface pasteurization technology Pulsed electric fields, including high-intensity Pulsed electric fields, including high-intensity
electric pulse (HELP)electric pulse (HELP) Ultrasonic energyUltrasonic energy High intensity visible lightHigh intensity visible light Ultraviolet lightUltraviolet light
Sao Paulo 2006Sao Paulo 2006 7474
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in foodGrowth and survivalGrowth and survival
Novel technologiesNovel technologies
Ghalfi et al. Bacteriocin activity by Lactobacillus curvatus CWBI-B28 to Ghalfi et al. Bacteriocin activity by Lactobacillus curvatus CWBI-B28 to inactivate Listeria monocytogenes in cold-smoked salmon during 4 degrees inactivate Listeria monocytogenes in cold-smoked salmon during 4 degrees C storage. C storage.
Dimitrijevic et al. Inhibitory effect of select nitrocompounds on growth and Dimitrijevic et al. Inhibitory effect of select nitrocompounds on growth and survivability of Listeria monocytogenes in vitro. survivability of Listeria monocytogenes in vitro.
Osaili et al. Thermal inactivation studies of Escherichia coli O157:H7, Osaili et al. Thermal inactivation studies of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in ready-to-eat chicken-fried beef Salmonella, and Listeria monocytogenes in ready-to-eat chicken-fried beef patties. patties.
Oussalah et al. Mechanism of action of Spanish oregano, Chinese cinnamon, Oussalah et al. Mechanism of action of Spanish oregano, Chinese cinnamon, and savory essential oils against cell membranes and walls of Escherichia and savory essential oils against cell membranes and walls of Escherichia coli O157:H7 and Listeria monocytogenescoli O157:H7 and Listeria monocytogenes. .
Sao Paulo 2006Sao Paulo 2006 7575
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in foodGrowth and survival of Listeria monocytogenes Growth and survival of Listeria monocytogenes
Microbiological Challenge TestingMicrobiological Challenge Testing
Challenge studyChallenge study
• Growth study – antimicrobial agentGrowth study – antimicrobial agent• Lethality study – post-lethality Lethality study – post-lethality
treatmenttreatment
Experienced food microbiologist, Experienced food microbiologist, understanding the growth limits of understanding the growth limits of LmLm
Sao Paulo 2006Sao Paulo 2006 7676
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in foodGrowth and survival of Listeria monocytogenes Growth and survival of Listeria monocytogenes
Microbiological Challenge TestingMicrobiological Challenge Testing
Determine if a study is usefulDetermine if a study is useful
• Study intrinsic properties of foodStudy intrinsic properties of food• Conduct microbiological growth modelingConduct microbiological growth modeling
Modified atmosphere packaged Modified atmosphere packaged products (vegetables, meats, poultry, products (vegetables, meats, poultry, fish) fish)
Sauces and salsas stored at ambient Sauces and salsas stored at ambient temperature temperature
Formula with new preservatives Formula with new preservatives Etc.Etc.
Sao Paulo 2006Sao Paulo 2006 7777
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in foodGrowth and survival of Listeria monocytogenes Growth and survival of Listeria monocytogenes
Microbiological Challenge TestingMicrobiological Challenge Testing
Factors that must be considered Factors that must be considered (Vestergaard, 2001) (Vestergaard, 2001)
1) the selection of appropriate pathogens or surrogates, 1) the selection of appropriate pathogens or surrogates,
2) the level of challenge inoculum, 2) the level of challenge inoculum,
3) the inoculum preparation and method of inoculation, 3) the inoculum preparation and method of inoculation,
4) the duration of the study, 4) the duration of the study,
5) formulation factors and storage conditions, and 5) formulation factors and storage conditions, and
6) sample analyses. 6) sample analyses.
Sao Paulo 2006Sao Paulo 2006 7878
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in foodGrowth and survival of Listeria monocytogenes Growth and survival of Listeria monocytogenes
Microbiological Challenge TestingMicrobiological Challenge Testing
Selection of challenge Selection of challenge organismsorganisms
3 – 5 strains associated with product and 3 – 5 strains associated with product and outbreakoutbreak
• L. monocytogenes L. monocytogenes for lab studies for lab studies• L. innocuaL. innocua for food processing facilities for food processing facilities
Resistance: Resistance: Listeria innocuaListeria innocua >> Lm Lm
Sao Paulo 2006Sao Paulo 2006 7979
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in foodGrowth and survival of Listeria monocytogenes Growth and survival of Listeria monocytogenes
Microbiological Challenge TestingMicrobiological Challenge Testing
Inoculum levelInoculum level
• Growth study: 100 – 1,000 cfu/gGrowth study: 100 – 1,000 cfu/g
• Post-lethality study: 10Post-lethality study: 104 4 – 10– 1066 cfu/g cfu/g
Sao Paulo 2006Sao Paulo 2006 8080
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in foodGrowth and survival of Listeria monocytogenes Growth and survival of Listeria monocytogenes
Microbiological Challenge TestingMicrobiological Challenge Testing
Inoculum preparation and method Inoculum preparation and method of inoculationof inoculation
• Stationary phase cells: Stationary phase cells: recontamination from environmental recontamination from environmental nichesniches
• Surface inoculation: vegetative, Surface inoculation: vegetative, slicedsliced
• Inoculum simulates intrinsic Inoculum simulates intrinsic properties of food: pH, water activity, properties of food: pH, water activity, salt level, etc.salt level, etc.
Sao Paulo 2006Sao Paulo 2006 8181
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in foodGrowth and survival of Listeria monocytogenes Growth and survival of Listeria monocytogenes
Microbiological Challenge TestingMicrobiological Challenge Testing
Duration of the study and Duration of the study and Formulation factors and storage Formulation factors and storage conditionsconditions
• Growth conditionsGrowth conditions
• NormalNormal• Moderate abuseModerate abuse• Worst-case scenarioWorst-case scenario
StorageStorage DistributionDistribution UseUse
Sao Paulo 2006Sao Paulo 2006 8282
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in foodGrowth and survival of Listeria monocytogenes Growth and survival of Listeria monocytogenes
Microbiological Challenge TestingMicrobiological Challenge Testing
Data interpretationData interpretation
The significance of a population increase The significance of a population increase varies with the hazard characterization varies with the hazard characterization
Low numbers of Low numbers of L. monocytogenesL. monocytogenes present a present a low risk to public healthlow risk to public health
Products that support the growth of the Products that support the growth of the microorganism present an increased riskmicroorganism present an increased risk
A 1 log increase is considered an A 1 log increase is considered an appropriate level of control for appropriate level of control for L. L. monocytogenesmonocytogenes
Sao Paulo 2006Sao Paulo 2006 8383
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in foodFinalFinal
Listeria monocytogenesListeria monocytogenes: a well studied : a well studied pathogen pathogen
RESEARCH NEEDSRESEARCH NEEDS
• DETECTION:DETECTION: identify its presence identify its presence more rapidly , distinguish avirulent more rapidly , distinguish avirulent strainsstrains
• TRACING:TRACING: more information on more information on transient and resident strains to transient and resident strains to prevent contaminationsprevent contaminations
• GROWTH AND SURVIVAL:GROWTH AND SURVIVAL: more more options to control Lm in RTE foodsoptions to control Lm in RTE foods
Sao Paulo 2006Sao Paulo 2006 8484
Controlling Controlling Listeria monocytogenesListeria monocytogenes in food in food
Thank you for your Thank you for your attention. attention.
Muito obrigado pela Muito obrigado pela atenção! atenção!