Listeria monocytogens Mandeep Balhara
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Listeria monocytogens Mandeep Balhara

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  • Within the Cell Cytoplasm: <br /> 1. Bacteria migrate to the periphery of the cytoplasm forming elongated protrusions (filopods) that can be ingested by adjacent cells <br /> 2. Polymerization of host actin around the bacterium which help to propel it through the cytoplasm. <br /> 3. Spreads from cell to cell without directly contacting the extracellular environment <br /> 4. Humerol immunity of relatively little importance <br /> 5. Cell mediated immunity primary means of defense <br />

Listeria monocytogens Mandeep Balhara Listeria monocytogens Mandeep Balhara Presentation Transcript

  • Conventional Method for the Detection of Listeria spp. From Dairy Products
  • Introduction  L. monocytogenes was discovered by Murray et al. (1926)  The first confirmed isolations from infected sheep were made in 1929 by Gill  L. monocytogenes is a Gram-positive, non spore forming bacterium  Found in soil, decaying plants and food, and causes listeriosis in animals and humans
  • The genus Listeria  The genus Listeria contains seven species: Pathogenic – L. monocytogenes pathogen – L. ivanovii - Non-pathogenic – – – – – L. L. L. L. L. innocua seeligeri welshimeri grayi murrayi - Human and Animal pathogen animal
  • The disease Listeriosis  Gastrointestinal Form – Organism causes damage to the absorptive villi affecting absorption of nutrients and promoting fluid secretion  Systemic Listeriosis – The organism passes through the intestinal barrier reaching the blood circulation and the lymphatic system  Abortion and Neonatal Listeriosis – L. monocytogenes crosses the placental barrier infects the fetus results in intrauterine infection and
  • Cell-cell transmission Entry Bacteria Internalins phagolysosome Fusion with another cell Escape 1. listeriolysin O 2. Phosphatidylinositolspecific phospholipase C replication actin tail Movement through cell Extrusion via filopods
  • Statistics in Developed Statistics in Developed Countries Countries     About 0.2 to 0.8 cases of listeriosis per 100,000 persons occur annually (Gellin et al., 1991; Lukinmaa et al., 2003) This results in 1600 to 8400 cases in Europe per year with 320 to 2500 death About 2,500 people in the U.S develop Listeriosis each year ( Mead et al., 1999) 5 out of every 100 people carry L. monocytogenes in their intestines  About 20 – 30 % of people die from the infection Source (DBMD)  L. monocytogenes reached the blood and cerebrospinal fluid in 89% of cases   Pregnant Death Rate women account for 27% of cases, immunodeficiency disorders account for 70% of cases. Extrapolation people with AIDS patients are 280 time more likely to contract Listeriosis than 500 per year, 41 per month, 9 per week, 1 per day others
  • Plant environment: Can colonize, multiply persist, attach and form biofilms Major concern: post-processing Contamination Possible Sources of Contamination Animals can contaminate foods of animal origin such as meats and dairy products Environmental sources: drains, conveyor belts, floor mats, foot baths, freezers, coolers, equipment, chilling rooms, cutting rooms, hands, packaging
  • Growth Parameter  Temperatu re Growth range = 30 to 113°F (-1 to 45°C) – Optimum = 86 to 98.6°F (30 to 37°C) – LM can survive freezing  Salt concentration – Growth at 20% – Survival at 25.5% – The organism can survive concentration of up to 16 % for a Acidity  Typical pH range is 5.0 to 9.6 – Optimum =neutral conditions ~6.0 - 7.0 year in a
  • Conventional cold enrichment method for isolation of Conventional cold enrichment method for isolation of Listeria spp. from food products Listeria spp. from food products Food sample is inoculated into a nutrient Broth without selective agents and held at 4°C for long periods After 24 h, and once a week, portions of the enrichment broth were plated onto selective media which were incubated at 35°C  Incubation at 4°C suppresses the growth of most microorganisms, but Listeria spp. multiply slowly with a generation time of 1.5 days Disadvantage  The need for prolonged incubation (up to several months or even a year) is a serious disadvantage
  • General scheme for the isolation of Listeria spp. from food products
  • ISO 11290-1/AFNOR Standard Method for the Isolation and Detection of Listeria spp. from dairy products 1 st Enrichment step Add 25 g of the sample to 225 ml of ½ Fraser broth Incubate at 30 0 C for 24 hr 2 nd Enrichment step Add 0.1 ml to 10 ml Fraser broth Incubate at 37 0 C for 4650 hr Streak on PALCAM / Oxford Agar Reading After 24-48 hr Streak on PALCAM / Oxford Agar Reading After 24-48 hr Drawback - Laborious Time consuming
  • Diagram of Procedure acc. to ISO/CD* draft 11290 and AFNOR* Detection of Listeria monocytogenes
  • Grams staining
  • Catalase test
  • Motility test
  • Rhamnose:(+)
  • CAMP test
  • Palcam Agar Base - Enumeration Principle and Interpretation • Differentiation on PALCAM Agar Base is based on Esculin hydrolysis and Mannitol fermentation • Listeria spp. hydrolyzes esculin, which appears as blackening in the medium • Mannitol and the pH indicator Phenol Red • Added to differentiate mannitol-fermenting strains of possible contaminants, including enterococci and staphylococci and appaer as yellow colonies • Listeria spp do not ferment Mannitol and appaer as olive green color colonies • Polymyixin B, Acriflavin, Ceftazidime, and Lithium Chloride are selective agents used to suppress Gramnegative and certain Gram-positive bacteria
  • Substrate- Esculin + = + Combines with Fe to give brown/black color Esculin + H2O = beta-D-glucose dihyroxycoumarin + 6,7-
  • Chromocult Listeria Selective Agar Base - Enumeration Principle and Interpretation  The rich basis of Chromocult Listeria Selective Agar is the addition of 5-bromo-4-chloro-3-indolyl-ß-Dglucopyranoside which makes it possible to differentiate between ß -D-glucosidase positive and negative bacteria  Listeriae are ß -D-glucosidase-positive and grow on the medium in the form of blue-green colonies  To detect L. Monocytogenes substrate -phosphatidylinositol is added to the medium  L. monocytogenes has the enzyme phosphatidylinositol phospholipase C (PI-PLC )  This phospholipase activity results in the formation of opaque haloes around L. monocytogenes colonies L- ß
  • Colony characteristics on Chromocult Listeria Selective Agar Base
  • Enumeration Principle  Enumeration of Listeria spp. is based on the principle that the organism show tolerance to the selective agent used in the isolation procedure  Ability to hydrolyse esculin by the enzyme betaglucosidase  Organism show blood agar  Ability to produce phosphatidylinositol-specific phospholipase-C (PI-PLC)  Ability to ferment rhamnose  So based on various formulation and combination different chromogenic and fluorogenic culture media have been developed weak beta-haemolysis on sheep
  • II) Development of Selective Liquid Medium (SLM) II) Development of Selective Liquid Medium (SLM) Screening of selective broths for enzymes activity by growing Listeria spp. and other potential contaminants Sr No Name of the Broth 1 Listeria Enrichment Broth (LEB) 2 Fraser Broth (FB) 3 University of Vermont Medium (UVM) 4 Brian Heart Infusion Broth (BHI) Marker Enzymes Color reaction at 37 o C ß-glucosidase Black (PI-PLC) α-d- mannosidase Blue Yellow Supplementation with Esculin and Ferric Ammonium Citrate results in blackening of broth or with Chromogenic substrates results in blue and yellow coloration
  • Chromogenic substrate For beta-glucosidase enzyme  5-Bromo-4 chloro3-indolyl- β -D glucopyranoside (X-GLU)  PNPG For α -mannosidase enzyme  p-nitrophenyl a lpha -mannoside For PI-PLC enzyme  5-Bromo-4-chloro-3-indoxyl myo-inositol-1 phosphate  For beta-glucosidase enzyme  4-methylumbelliferyl β -D glucoside  For alpha-D-mannosidase enzyme  4-methylumbelliferyl-alpha-D-mannopyranoside  For PI-PLC enzyme  4-Methylumbelliferyl myo-inositol-1-phosphate
  • Incubation at 37 o C Incubation at 37 o C
  • Selective agent used in the isolation of Listeria spp. Antimicrobial Agent mg/L Organism inhibited Acriflavin 5-25 gram-positives, including Lactobacillus bulgaricus and Streptococcus thermophilus Nalidixic acid 20-40 gram-negatives except Pseudomonas and Proteus Moxalactam, phenylethanol 20 Gram-negative bacteria Ceftazidime 50 Gram-negative bacteria Cycloheximide 50 Fungi Fosfomycin 100 Inhibitory to Staphylococcus and Bacillus
  • Pure cells of Pure cells of Listeria spp. Listeria spp. Spiked milk Spiked milk samples samples Natural milk Natural milk Grown in SLM under optimised conditions
  • Thank you