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São Paulo 2006. Controlling Listeria monocytogenes in food Joaquín Martínez Suárez Departamento de Tecnología de Alimentos INIA Madrid. INIA Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria. National Institute for Agriculture and Food Research and Technology.

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S o paulo 2006

São Paulo 2006

Controlling

Listeria monocytogenes in food

Joaquín Martínez Suárez

Departamento de Tecnología de Alimentos

INIA

Madrid

Sao Paulo 2006


S o paulo 2006

INIAInstituto Nacional de Investigación y Tecnología Agraria y Alimentaria

National Institute for Agriculture and Food Research and Technology


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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


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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: 35Funding: 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.

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Department of food technology

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

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Controlling listeria monocytogenes in food

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’

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Controlling listeria monocytogenes in food1

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

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Controlling listeria monocytogenes in food introduction to the organism

Controlling Listeria monocytogenes in foodIntroduction to the organism

  • Taxonomy

  • Ecology

  • Virulence

  • Listeriosis

  • Outbreaks

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


Introduction to the organism taxonomy grouping of listeria monocytogenes serotypes

Introduction to the organismTaxonomyGrouping of Listeria monocytogenes serotypes


Introduction to the organism ecology sources of listeria species

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

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Introduction to the organism virulence

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

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Introduction to the organism virulence1

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


Introduction to the organism listeriosis

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

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Introduction to the organism outbreaks

Introduction to the organismOutbreaks


Introduction to the organism outbreaks1

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

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Specific detection of listeria monocytogenes in foods

Specific detection of Listeria monocytogenes in foods

Sao Paulo 2006


Some commercial methods used for the specific detection or identification listeria monocytogenes

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


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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

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S o paulo 2006

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


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Alternative rapid methods for screening of enriched samples and/or culture confirmation

Specific detection of Listeria monocytogenes in foods

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Some commercial methods used for the specific detection or identification listeria monocytogenes1

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


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Specific detection of Listeria monocytogenes in foods Immunoassay methodsELFA (Enzyme Linked Fluorescent Assay)

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Specific detection of listeria monocytogenes in foods nucleic acid based methods dna hybridization

Specific detection of Listeria monocytogenes in foods Nucleic acid-based methodsDNA hybridization

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Some commercial methods used for the specific detection or identification listeria monocytogenes2

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


Specific detection of listeria monocytogenes in foods nucleic acid based methods pcr

Specific detection of Listeria monocytogenes in foods Nucleic acid-based methodsPCR

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Specific detection of listeria monocytogenes in foods nucleic acid based methods pcr1

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

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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


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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

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Specific detection of Listeria monocytogenes in foods Future trends of Listeria monocytogenes detection methods

Novel methods

  • Microarrays or biochips

  • Biosensors

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


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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)

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Monitoring for listeria monocytogenes in food plants and tracing of the organism

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

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Monitoring for listeria monocytogenes in food plants and tracing of the organism1

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

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Monitoring for listeria monocytogenes in food plants and tracing of the organism2

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

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Monitoring for listeria monocytogenes in food plants and tracing of the organism3

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

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Monitoring for listeria monocytogenes in food plants and tracing of the organism4

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

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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)


Monitoring for listeria monocytogenes in food plants and tracing of the organism5

Monitoring for Listeria monocytogenes in food plantsand tracing of the organism

FOOD SAMPLES

  • IN-PROCESS PRODUCT TESTING

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Monitoring for listeria monocytogenes in food plants and tracing of the organism6

Monitoring for Listeria monocytogenes in food plantsand tracing of the organism

FOOD SAMPLES

END-PRODUCT TESTING

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Monitoring for listeria monocytogenes in food plants and tracing of the organism7

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

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Monitoring for listeria monocytogenes in food plants and tracing of the organism8

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

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Monitoring for listeria monocytogenes in food plants and tracing of the organism9

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

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Monitoring for listeria monocytogenes in food plants and tracing of the organism10

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

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Monitoring for listeria monocytogenes in food plants and tracing of the organism11

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

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Monitoring for listeria monocytogenes in food plants and tracing of the organism12

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)

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Monitoring for listeria monocytogenes in food plants and tracing of the organism13

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

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Monitoring for listeria monocytogenes in food plants and tracing of the organism14

Monitoring for Listeria monocytogenes in food plantsand tracing of the organism

Listeria monocytogenes subtyping methods

PCR-serotyping (Doumith et al., 2004): targets and products


Monitoring for listeria monocytogenes in food plants and tracing of the organism15

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


Monitoring for listeria monocytogenes in food plants and tracing of the organism16

Monitoring for Listeria monocytogenes in food plantsand tracing of the organism

Listeria monocytogenes subtyping methods

Pulsed field gel electrophoresis (PFGE)

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Monitoring for listeria monocytogenes in food plants and tracing of the organism17

Monitoring for Listeria monocytogenes in food plantsand tracing of the organism

Listeria monocytogenes subtyping methods

Ribotyping

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Monitoring for listeria monocytogenes in food plants and tracing of the organism18

Monitoring for Listeria monocytogenes in food plantsand tracing of the organism

Listeria monocytogenes subtyping methods

Multilocus sequence typing (MLST)


Monitoring for listeria monocytogenes in food plants and tracing of the organism19

Monitoring for Listeria monocytogenes in food plantsand tracing of the organism

3 studies on poultry contamination:

  • Abattoir

  • Processing plant

  • Retail

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Monitoring for listeria monocytogenes in food plants and tracing of the organism20

Monitoring for Listeria monocytogenes in food plantsand tracing of the organism

Molecular subtyping of

Listeria monocytogenes originating from a broiler abattoir

Victoria López, Sagrario Ortiz, Alfredo Corujo, Pilar López, Jaime Navas, Raúl Moreno, Joaquín V. Martínez-Suárez

Sao Paulo 2006


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Monitoring for Listeria monocytogenes in food plantsand tracing of the organismMolecular subtyping of Listeria monocytogenes originating from a broiler abattoir Sampling results

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Monitoring for Listeria monocytogenes in food plantsand tracing of the organismMolecular subtyping of Listeria monocytogenes originating from a broiler abattoir Pulsed field gel electrophoresis (PFGE)

AscI

ApaI

S 1 2 4 5 6 S 7 3 8 9 S

S D A B S C H E J S

- 622 Kb

- 220 Kb

- 37 Kb


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Monitoring for Listeria monocytogenes in food plantsand tracing of the organismMolecular subtyping of Listeria monocytogenes originating from a broiler abattoir Asc I / Apa I pulsotypes


Monitoring for listeria monocytogenes in food plants and tracing of the organism21

Monitoring for Listeria monocytogenes in food plantsand tracing of the organism

Source of the contamination with Listeria monocytogenes serotype 4b in processed chicken products

Victoria López, Sagrario Ortiz, Alfredo Corujo, Pilar López, Jaime Navas, Raúl Moreno, and Joaquín V. Martínez-Suárez

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Monitoring for Listeria monocytogenes in food plantsand tracing of the organism Source of the contamination with Listeria monocytogenes serotype 4b in processed chicken productsDistribution of the isolates of L. monocytogenes found in different retail broiler products, August of 2005


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Monitoring for Listeria monocytogenes in food plantsand tracing of the organism Source of the contamination with Listeria monocytogenes serotype 4b in processed chicken productsSerotypes and PFGE profiles of the Listeria monocytogenes isolates from pork fat


Monitoring for listeria monocytogenes in food plants and tracing of the organism22

Monitoring for Listeria monocytogenes in food plantsand tracing of the organism

Listeria monocytogenes in poultry products

at retail

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

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Monitoring for Listeria monocytogenes in food plantsand tracing of the organism Listeria monocytogenes in poultry products at retail


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Monitoring for Listeria monocytogenes in food plantsand tracing of the organism Listeria monocytogenes in poultry products at retailApaI results


Controlling listeria monocytogenes in food growth and survival

Controlling Listeria monocytogenes in foodGrowth and survival

Preventing growth of Lm in contaminated ready-to-eat foods through time and temperature controls, or through reformulation of foods so that they retard or do not support growth of Lm

Sao Paulo 2006


Controlling listeria monocytogenes in food growth and survival1

Controlling Listeria monocytogenes in foodGrowth 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

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Controlling Listeria monocytogenes in foodGrowth and survivalLimits for the growth of Listeria monocytogenes (ICMSF, 1996)


Controlling listeria monocytogenes in food growth and survival2

Controlling Listeria monocytogenes in foodGrowth and survival

Products not capable of supporting the growth of Lm

Water activity (Aw) value of less than 0.92

pH less than 4.39 when measured at 24ºC

Food in an unopened, sealed container that is commercially sterile under non-refrigerated storage

Laboratory evidence demonstrates that no growth of Lm can occur

The product does not support the growth of any microorganisms

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Controlling Listeria monocytogenes in foodGrowth and survival of Listeria monocytogenes Stress responsesHeat-shock response and acid adaptation

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Controlling listeria monocytogenes in food growth and survival3

Controlling Listeria monocytogenes in foodGrowth and survival

Combining factors

Other means for controlling Lm

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Controlling listeria monocytogenes in food growth and survival4

Controlling Listeria monocytogenes in foodGrowth and survival

Other means for controlling Lm

Currently used chemicals

  • Sodium hypochlorite

  • Gas atmosphere

  • Sodium nitrite

  • Other antimicrobial chemicals (sodium lactate, sodium acetate, sodium diacetate, sodium benzoate, sodium propionate, potassium sorbate)

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Controlling listeria monocytogenes in food growth and survival5

Controlling Listeria monocytogenes in foodGrowth and survival

Other means for controlling Lm

Currently used nonthermal technologies

  • Nisin and other bacteriocins

  • Gamma irradiation

  • High-hydrostatic-pressure technology

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Controlling listeria monocytogenes in food growth and survival6

Controlling Listeria monocytogenes in foodGrowth and survival

Other means for controlling Lm

Novel technologies: Chemical products

  • Hydrogen peroxide

  • Calcinated calcium

  • Plant essential oils

  • Ozone

  • Acidic calcium sulfate, lactic acid and propionic acid

  • Peracetic acid (15%) and H2O2 (10%)

  • Peroxyacetic acid-based additive

  • Sodium or potassium lactate and sodium diacetate

  • Chlorine dioxide

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Controlling listeria monocytogenes in food growth and survival7

Controlling Listeria monocytogenes in foodGrowth and survival

Other means for controlling Lm

Novel technologies:Physical treatments

  • Surface pasteurization technology

  • Pulsed electric fields, including high-intensity electric pulse (HELP)

  • Ultrasonic energy

  • High intensity visible light

  • Ultraviolet light

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Controlling listeria monocytogenes in food growth and survival8

Controlling Listeria monocytogenes in foodGrowth and survival

Novel technologies

Ghalfi et al. Bacteriocin activity by Lactobacillus curvatus CWBI-B28 to inactivate Listeria monocytogenes in cold-smoked salmon during 4 degrees C storage.

Dimitrijevic et al. Inhibitory effect of select nitrocompounds on growth and survivability of Listeria monocytogenes in vitro.

Osaili et al. Thermal inactivation studies of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in ready-to-eat chicken-fried beef patties.

Oussalah et al. Mechanism of action of Spanish oregano, Chinese cinnamon, and savory essential oils against cell membranes and walls of Escherichia coli O157:H7 and Listeria monocytogenes.

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Controlling Listeria monocytogenes in foodGrowth and survival of Listeria monocytogenes Microbiological Challenge Testing

Challenge study

  • Growth study – antimicrobial agent

  • Lethality study – post-lethality treatment

    Experienced food microbiologist, understanding the growth limits of Lm

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Controlling Listeria monocytogenes in foodGrowth and survival of Listeria monocytogenes Microbiological Challenge Testing

  • Determine if a study is useful

    • Study intrinsic properties of food

    • Conduct microbiological growth modeling

      • Modified atmosphere packaged products (vegetables, meats, poultry, fish)

      • Sauces and salsas stored at ambient temperature

      • Formula with new preservatives

      • Etc.

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Controlling Listeria monocytogenes in foodGrowth and survival of Listeria monocytogenes Microbiological Challenge Testing

Factors that must be considered

(Vestergaard, 2001)

1) the selection of appropriate pathogens or surrogates,

2) the level of challenge inoculum,

3) the inoculum preparation and method of inoculation,

4) the duration of the study,

5) formulation factors and storage conditions, and

6) sample analyses.

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Controlling Listeria monocytogenes in foodGrowth and survival of Listeria monocytogenes Microbiological Challenge Testing

Selection of challenge organisms

  • 3 – 5 strains associated with product and outbreak

    • L. monocytogenes for lab studies

    • L. innocua for food processing facilities

      • Resistance: Listeria innocua> Lm

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Controlling Listeria monocytogenes in foodGrowth and survival of Listeria monocytogenes Microbiological Challenge Testing

  • Inoculum level

    • Growth study: 100 – 1,000 cfu/g

    • Post-lethality study: 104 – 106 cfu/g

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Controlling Listeria monocytogenes in foodGrowth and survival of Listeria monocytogenes Microbiological Challenge Testing

Inoculum preparation and method of inoculation

  • Stationary phase cells: recontamination from environmental niches

  • Surface inoculation: vegetative, sliced

  • Inoculum simulates intrinsic properties of food: pH, water activity, salt level, etc.

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Controlling Listeria monocytogenes in foodGrowth and survival of Listeria monocytogenes Microbiological Challenge Testing

Duration of the study and Formulation factors and storage conditions

  • Growth conditions

    • Normal

    • Moderate abuse

    • Worst-case scenario

      • Storage

      • Distribution

      • Use

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Controlling Listeria monocytogenes in foodGrowth and survival of Listeria monocytogenes Microbiological Challenge Testing

Data interpretation

The significance of a population increase varies with the hazard characterization

Low numbers of L. monocytogenes present a low risk to public health

Products that support the growth of the microorganism present an increased risk

A 1 log increase is considered an appropriate level of control for L. monocytogenes

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Controlling listeria monocytogenes in food final

Controlling Listeria monocytogenes in foodFinal

Listeria monocytogenes: a well studied pathogen

RESEARCH NEEDS

  • DETECTION: identify its presence more rapidly , distinguish avirulent strains

  • TRACING: more information on transient and resident strains to prevent contaminations

  • GROWTH AND SURVIVAL: more options to control Lm in RTE foods

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Controlling listeria monocytogenes in food2

Controlling Listeria monocytogenes in food

Thank you for your attention.

Muito obrigado pela atenção!

Sao Paulo 2006


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