S

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São Paulo 2006

• Controlling
• Listeria monocytogenes in food
• Joaquín Martínez Suárez
• Departamento de Tecnología de Alimentos
• INIA
• Madrid

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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 35 Funding 310.000

• Research lines
• Organoleptic quality and directed maturation of
  Spanish cheese through enzymatic and
  microbiological processes.
• Processing and nutritional quality in Vegetable
  products.
• Composition and quality in lamb meat.
• Food microbiological safety using biological
  systems.

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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

• 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 food

• Introduction to the organism
• Specific detection of Listeria monocytogenes in
  foods
• Monitoring for Listeria monocytogenes and tracing
  of the organism in food plants
• Growth and survival of Listeria monocytogenes and
  microbiological challenge studies

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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

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Introduction to the organismTaxonomyGrouping
of Listeria monocytogenes serotypes
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Introduction to the organismEcologySources of
Listeria species

• faeces (wild and domestic animals)
• humans
• mammals
• birds
• fish
• shellfish

• water
• soil
• sewage
• decaying vegetation
• silage
• plants (cultivated and uncultivated)

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Introduction to the organismVirulence

• FACTORS
• host immunity
• number of cells ingested
• virulence of the specific strain

• HOST CELL INFECTION
• invasion
• multiplication with the hosts cell cytoplasm
• polymerization of host cell actin
• movement across the hosts cell cytoplasm
• invasion of adjacent cells

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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

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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 organismOutbreaks
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Introduction to the organismOutbreaks

• 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 (gt10 days)
• Food is ready-to-eat
• Consumption by vulnerable groups

• Food
• Amount and frequency of consumption
• Frequency and level of contamination

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Specific detection of Listeria monocytogenes in
foods
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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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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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Specific detection of Listeria monocytogenes
in foods

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

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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 methodsDNA
hybridization
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Some commercial methods used for the specific
detection or identification Listeria
monocytogenes
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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 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 lt4-5 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

• 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 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 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 organism

• FOOD SAMPLES
• incoming raw materials

• Harbor Lm
• raw, commercially available ground meat
• broiler carcasses
• turkey carcasses
• cow and bull carcasses
• hog carcasses

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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)
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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
plantsand tracing of the organism

• FOOD SAMPLES
• END-PRODUCT TESTING

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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
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
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
plantsand tracing of the organism

• ENVIRONMENTAL SAMPLING
• NON-FOOD CONTACT SURFACES

• Common reservoirs
• Floors
• Drains
• Walls
• Ceilings
• Overhead structures
• Catwalks
• Insulation
• Cleaning tools
• Maintenance tools
• Equipment framework
• Condensate

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Monitoring for Listeria monocytogenes in food
plantsand tracing of the organism

• 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

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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
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
plantsand tracing of the organism

• Listeria monocytogenes subtyping methods
• PCR-serotyping (Doumith et al., 2004) targets
  and products

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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

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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
plantsand tracing of the organism

• Listeria monocytogenes subtyping methods
• Ribotyping

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Monitoring for Listeria monocytogenes in food
plantsand tracing of the organism

• Listeria monocytogenes subtyping methods
• Multilocus sequence typing (MLST)

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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
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

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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
Month Nº Lm 1/2a 1/2b
March 10 2 (non-contact) 8
April 19 1 (non-contact) 18
May 20 2 (non-contact) 18
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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

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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
  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 products
Distribution 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 products
Serotypes and PFGE profiles of the Listeria
monocytogenes isolates from pork fat
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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
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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

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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 survival Limits for the growth
of Listeria monocytogenes (ICMSF, 1996)
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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
foodGrowth and survival

• Combining factors
• Other means for controlling Lm

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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
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
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
foodGrowth and survival

• Other means for controlling Lm
• Novel technologiesPhysical 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
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 O157H7, 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 O157H7 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 gt 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 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 food

• Thank you for your attention.
• Muito obrigado pela atenção!