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Listeria monocytogenes. Listeria monocytogenes. Invasive pathogen WEAR GLOVES!!! Listeriosis YOPI Miscarriage, stillbirth, premature birth Bacteremia Meningitis Meningoencephalitis. Location of L. monocytogenes. Ubiquitous Raw foods Processed RTE foods Food processing environment.

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Listeria monocytogenes


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listeria monocytogenes2
Listeria monocytogenes
  • Invasive pathogen
  • WEAR GLOVES!!!

Listeriosis

  • YOPI
  • Miscarriage, stillbirth, premature birth
  • Bacteremia
  • Meningitis
  • Meningoencephalitis
location of l monocytogenes
Location of L. monocytogenes
  • Ubiquitous
  • Raw foods
  • Processed RTE foods
  • Food processing environment
characteristics of l monocytogenes
Characteristics of L. monocytogenes
  • Gram-positive, non-sporeformer
  • Oxidase negative, Catalase positive
  • Psychrotrophic
  • Salt tolerant
  • Microaerophilic
  • Tumbling motility
  • Weak β-hemolysis
  • Hydrolyzes esculin
  • CAMP ++
  • Sugar fermentation: xylose (-), rhamnose (+), mannitol (-)
identification in foods
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

media
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.
media continued
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
media continued9
Media (continued)
  • Identification: Tryptic Soy agar with blood (TSA-blood)
    • Cultivation of fastidious organisms
    • Differential
      • Hemolysis under high CO2
        • α-hemolysis
          • Greenish discoloration around colony
        • β-hemolysis
          • Clear zone around colony (TRUE HEMOLYSIS)
        • γ-hemolysis
          • No change around colony
  • Identification: Tryptic Soy agar with yeast extract (TSAYE)
    • Nonselective
    • Colony morphology
    • Catalase reaction
overview of procedures
Overview of Procedures

Food/Environmental Sample

1

Incubation

Enrichments

UVM1

Transfer

2

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

3

Cell lysis: heat at 37°C-30 min, add proteinase K and Buffer AL, then 70°C-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

4

Gel with DNA bands

Electrophoresis

Identify DNA band that corresponds to Listeria spp.

culturing and dna isolation
Culturing and DNA Isolation
  • Primary enrichment and selective enrichment same as conventional
  • DNA isolation using Qiagen DNeasy kit protocol
  • DNA used for PCR
pcr overview
PCR Overview

Need: DNA polymerase (Taq), reaction buffer containing Mg, dNTPs, oligonucleotide primers, water, and DNA sample

gene of interest
Gene of interest
  • iap gene
    • Invasion Associated Protein – p60
    • Conserved in all Listeria spp.
  • PCR product
    • 1.5 kb
    • Visualize by agarose gel electrophoresis
agarose gel electrophoresis
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
rapid method result
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

overall results
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

Overall Results

Both conventional and rapid methods are specific to the genus level

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

changes to procedure
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