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Listeria monocytogenes

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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  1. Listeria monocytogenes

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

  3. Location of L. monocytogenes • Ubiquitous • Raw foods • Processed RTE foods • Food processing environment

  4. 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 (-)

  5. 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

  6. Conventional Method

  7. 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.

  8. 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

  9. 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

  10. Rapid Method

  11. 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.

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

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

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

  15. 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

  16. 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

  17. 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.

  18. 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

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