Listeria monocytogenes

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Listeria monocytogenes
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Listeria monocytogenes

• Invasive pathogen
• WEAR GLOVES!!!
• Listeriosis
• YOPI
• Miscarriage, stillbirth, premature birth
• Bacteremia
• Meningitis
• Meningoencephalitis

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Location of L. monocytogenes

• Ubiquitous
• Raw foods
• Processed RTE foods
• Food processing environment

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Characteristics of L. monocytogenes

• Gram-positive, non-sporeformer
• Oxidase negative, Catalase positive
• Psychrotrophic
• Salt tolerant
• Microaerophilic
• Tumbling motility
• Weak ß-hemolysis
• Hydrolyzes esculin
• CAMP
• Sugar fermentation xylose (-), rhamnose (),
  mannitol (-)

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Identification in foods

• Detection is more important than counting
• A Conventional Method
• Culturing on various selective and differential
  media (enrichment, isolation, biochemical
  analysis)
• B Rapid Method
• Genetic based PCR method

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Conventional Method
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Media

• Primary Enrichment UVM1 broth
• Selective agents
• Nalidixic acid inhibits Gram-negative
• Acriflavin suppresses most other Gram-positive
• Selective Enrichments Fraser broth
• Selective agents
• Nalidixic acid and Acriflavin
• Lithium Chloride inhibits enterococci
• Differential agents
• Esculin and Ferric ammonium citrate (FAC)
  esculin hydrolysis in the presence of ferric ions
  black ppt.

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Media (continued)

• Isolation Modified Oxford agar (MOX)
• Selective agents
• Colistin sulfate, Moxalactam, lithium chloride
• Differential agents
• Esculin and FAC
• Isolation PALCAM agar
• Selective agents
• Lithium chloride, polymyxin, acriflavin,
  ceftazidine
• Differential agents
• Esculin and FAC
• Mannitol and phenol red
• Listeria spp. will appear black with red
  surrounding

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Media (continued)

• Identification Tryptic Soy agar with blood
  (TSA-blood)
• Cultivation of fastidious organisms
• Differential
• Hemolysis under high CO2
• a-hemolysis
• Greenish discoloration around colony
• ß-hemolysis
• Clear zone around colony (TRUE HEMOLYSIS)
• ?-hemolysis
• No change around colony
• Identification Tryptic Soy agar with yeast
  extract (TSAYE)
• Nonselective
• Colony morphology
• Catalase reaction

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Rapid Method
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Overview of Procedures
Food/Environmental Sample
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Incubation
Enrichments
UVM1
Transfer
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Positive and negative controls(L. monocytogenes
and E. faecalis)
FraserBroth
Incubation
Transfer into microcentrifuge tubes. Mix with
lysis buffer.
Laboratory Period
DNA extraction
microcentrifuge tube
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Cell lysis heat at 37C-30 min, add proteinase K
and Buffer AL, then 70C-30 min
Continue with DNA isolation using DNeasy spin
column.
PCR Reaction mixture (Water, Reaction Buffer,
primers, dNTPs, Taq)
PCR tube
PCR
Run the thermocycler. Cool or refrigerate.
Gel electrophoresis
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Gel with DNA bands
Electrophoresis
Identify DNA band that corresponds to Listeria
spp.
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Culturing and DNA Isolation

• Primary enrichment and selective enrichment same
  as conventional
• DNA isolation using Qiagen DNeasy kit protocol
• DNA used for PCR

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PCR Overview
Need DNA polymerase (Taq), reaction buffer
containing Mg, dNTPs, oligonucleotide primers,
water, and DNA sample
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Gene of interest

• iap gene
• Invasion Associated Protein p60
• Conserved in all Listeria spp.
• PCR product
• 1.5 kb
• Visualize by agarose gel electrophoresis

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Agarose Gel Electrophoresis

• Agarose is a linear polymer (sieve)
• Rate of migration dependent on size and charge
• DNA is negatively charged
• Larger molecules move slower
• DNA ladder size measurement
• Loading dye - visualization
• Ethidium bromide (MUTAGEN) binds to DNA,
  fluoresces under UV

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Rapid Method Result

• Gel picture

100 bp DNA ladder
L. monocytogenes
L. monocytogenes
L. innocua
L. innocua
E. faecalis
E. faecalis
1.5 Kb bp band
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Overall Results
Both conventional and rapid methods are specific
to the genus level

• Rapid Method
• 1.5 kb band Listeria ()

• Conventional method
• Gram reaction purple ()
• Catalase bubbles ()
• Fraser black
• MOX black colonies
• PALCAM black colonies, red agar
• TSA-blood variable
• TSAYE blue-gray tint

Further characterization may be completed with
API strips, CAMP test, DNA sequencing, etc.
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Changes to Procedure

• Each pair has one sample
• As a group of four you will have both a positive
  and negative control
• As a group of four you will have a total of four
  samples (2 food, 2 controls ( and -)
• After centrifuging Fraser broth sample, pipet off
  the supernatant