São Paulo 2006

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


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.


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


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’



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




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


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



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


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)


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


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



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


Chromogenic media Chromogenic media

Evaluation of ALOA plating medium for its suitabilityto recover high pressure-injured Listeria monocytogenes

from ground chicken meat



Alternative rapid methods for Alternative rapid methods for screening of enriched samples screening of enriched samples

and/or culture confirmationand/or culture confirmation


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)


Specific detection of Specific detection of Listeria monocytogenesListeria monocytogenes in foods in foods Nucleic acid-based methodsNucleic acid-based methods

DNA hybridizationDNA hybridization



PCRPCR



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


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)


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


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


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


2-4 Screening, food and environmental

samples

107

Real-time PCR 2 Screening, food 105



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)


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



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



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


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


Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food in food plants and tracing of the organismplants and tracing of the organism


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


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


Monitoring for Monitoring for Listeria monocytogenesListeria monocytogenes in food plants in food plantsand tracing of the organismand tracing of the organism


IN-PROCESS IN-PROCESS PRODUCT TESTINGPRODUCT TESTING




END-PRODUCT END-PRODUCT TESTINGTESTING



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




sampling to minimize sampling to minimize environmental environmental contamination with Lmcontamination with Lm

effective plan to take effective plan to take corrective actioncorrective action



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




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




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



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)



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


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.



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



Listeria monocytogenesListeria monocytogenes subtyping methodssubtyping methods

Pulsed field gel Pulsed field gel electrophoresis (PFGE)electrophoresis (PFGE)




RibotypingRibotyping



Multilocus sequence typing (MLST)Multilocus sequence typing (MLST)



3 studies on poultry 3 studies on poultry contamination:contamination:

AbattoirAbattoir Processing plantProcessing plant RetailRetail



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



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



Pulsed field gel electrophoresis (PFGE)Pulsed field gel electrophoresis (PFGE)



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


P1

P2

P3

P4

P5

P6

P7

P8

P9

P10

P11

P12

0

1

2

3

4

5

6

7

8




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


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



Navas, J., Ortiz, S., López, P., López, V., Martínez-Suárez, J.V.

Listeria monocytogenesListeria monocytogenes in poultry products in poultry products

at retailat retail


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


Listeria monocytogenesListeria monocytogenes in poultry products at retail in poultry products at retail

ApaApaI resultsI results



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




Factors affecting the growth and survival of Lm

•temperature•pH•water activity

Products not capable of supporting the growth of Lm

Stress responses



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 -


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


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




Combining factorsCombining factors

Other means for Other means for controlling Lmcontrolling Lm




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)





Currently used nCurrently used nonthermal technologiesonthermal technologies

Nisin and other bacteriocinsNisin and other bacteriocins Gamma irradiationGamma irradiation High-hydrostatic-pressure technologyHigh-hydrostatic-pressure technology





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





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


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. .



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




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.




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.




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




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




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.




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




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


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



Thank you for your Thank you for your attention. attention.

Muito obrigado pela Muito obrigado pela atenção! atenção!

São Paulo 2006

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Transcript of São Paulo 2006