Conventional Method for the Conventional Method for the Detection of Detection of Listeria Listeria spp. From spp. From

Dairy ProductsDairy Products

L. monocytogenes L. monocytogenes was discovered by was discovered by Murray et al. (1926)Murray et al. (1926)

The first confirmed isolations from infected sheep were The first confirmed isolations from infected sheep were made in made in 1929 1929 by by Gill Gill

L. monocytogenes L. monocytogenes is a Gram-positive, non spore forming is a Gram-positive, non spore forming bacteriumbacterium

Found in soil, decaying plants and food, and causes Found in soil, decaying plants and food, and causes listeriosis in animals and humanslisteriosis in animals and humans

Introduction Introduction

The genus Listeria contains seven species:

Pathogenic

– L. monocytogenes - Human and animal pathogen

– L. ivanovii - Animal pathogen

Non-pathogenic– L. innocua– L. seeligeri – L. welshimeri – L. grayi – L. murrayi

The genus Listeria

Gastrointestinal FormGastrointestinal Form– Organism causes damage to the absorptive villi Organism causes damage to the absorptive villi

affecting absorption of nutrients and promoting affecting absorption of nutrients and promoting fluid secretionfluid 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 and

infects the fetus results in intrauterine infection

The disease Listeriosis

Cell-cell transmissionCell-cell transmission

Fusion with Fusion with another cellanother cell

EntryEntryInternalinsInternalins

EscapeEscape1. listeriolysin O1. listeriolysin O

2. Phosphatidylinositol-2. Phosphatidylinositol-specific phospholipase Cspecific phospholipase C

actin tailactin tailMovement through cellMovement through cell

Extrusion via Extrusion via

filopodsfilopodsreplicationreplication

BacteriaBacteria

phagolysosomephagolysosome

Death Rate Extrapolation

About 0.2 to 0.8 cases of listeriosis per 100,000 persons occur About 0.2 to 0.8 cases of listeriosis per 100,000 persons occur annuallyannually

(Gellin et al., 1991; Lukinmaa (Gellin et al., 1991; Lukinmaa et al.,et al., 2003) 2003)

This results in 1600 to 8400 cases in Europe per year with 320 to 2500 This results in 1600 to 8400 cases in Europe per year with 320 to 2500 deathdeath

About 2,500 people in the U.S develop About 2,500 people in the U.S develop ListeriosisListeriosis each year each year ((Mead Mead et al.,et al.,

1999)1999) 5 out of every 100 people carry 5 out of every 100 people carry L. monocytogenesL. monocytogenes in their intestines in their intestines

About 20 – 30 % of people die from the infectionAbout 20 – 30 % of people die from the infection Source (DBMD)Source (DBMD)

L. monocytogenesL. monocytogenes reached the blood and cerebrospinal fluid in 89% reached the blood and cerebrospinal fluid in 89% of casesof cases

Pregnant women account for 27% of cases, people with Pregnant women account for 27% of cases, people with immunodeficiency disorders account for 70% of cases.immunodeficiency disorders account for 70% of cases.

AIDS patients are 280 time more likely to contract AIDS patients are 280 time more likely to contract ListeriosisListeriosis than than othersothers

Statistics in Developed Countries

Statistics in Developed Countries

500 per year, 41 per month, 9 per week, 1 per day

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

Plant environment: Plant environment: Can colonize, multiply Can colonize, multiply

persist, attach and form biofilmspersist, attach and form biofilms

Major concern: Major concern: post-processing post-processing ContaminationContamination

Growth Parameter Growth Parameter

Growth range = 30 to 113°F (-1 to 45°C) Growth range = 30 to 113°F (-1 to 45°C) – Optimum = 86 to 98.6°F (30 to 37°C)Optimum = 86 to 98.6°F (30 to 37°C)

– LM can survive freezingLM can survive freezing

Salt concentrationSalt concentration– Growth at 20%Growth at 20%– Survival at 25.5% Survival at 25.5% – The organism can survive for a year in a The organism can survive for a year in a

concentration of up to 16 %concentration of up to 16 %

TemperatuTemperaturere

Typical pH range is 5.0 to 9.6Typical pH range is 5.0 to 9.6– Optimum =neutral conditions ~6.0 - 7.0Optimum =neutral conditions ~6.0 - 7.0

AcidityAcidity

Incubation at 4°C suppresses the growth of most Incubation at 4°C suppresses the growth of most microorganisms, but microorganisms, but ListeriaListeria spp. multiply slowly with a spp. multiply slowly with a generation time of 1.5 daysgeneration time of 1.5 days

The need for prolonged incubation (up to several months or The need for prolonged incubation (up to several months or even a year) is a serious disadvantageeven a year) is a serious disadvantage

Disadvantage Disadvantage

Conventional cold enrichment method for isolation of Conventional cold enrichment method for isolation of Listeria spp.Listeria spp. from food products from food products

Conventional cold enrichment method for isolation of Conventional cold enrichment method for isolation of Listeria spp.Listeria spp. from food products 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

General scheme for the isolation of General scheme for the isolation of ListeriaListeria spp. from spp. from food products food products

ISO 11290-1/AFNOR StandardISO 11290-1/AFNOR Standard Method for the Isolation and Method for the Isolation and Detection of Detection of ListeriaListeria spp. from dairy products spp. from dairy products

Add 25 g of the sample to 225 ml of ½ Add 25 g of the sample to 225 ml of ½ Fraser broth Fraser broth

11stst Enrichment Enrichment stepstep

Incubate at 30Incubate at 3000C for 24 C for 24 hrhr

Add 0.1 ml to 10 ml Fraser Add 0.1 ml to 10 ml Fraser brothbroth

Incubate at 37Incubate at 3700C for 46-C for 46-50 hr50 hr

Streak on Streak on PALCAM / PALCAM /

Oxford Oxford AgarAgar

22ndnd Enrichment Enrichment stepstep

Drawback - Laborious Drawback - Laborious Time consuming Time consuming

Streak on Streak on PALCAM / PALCAM /

Oxford Oxford AgarAgar

Reading After 24-48 hrReading After 24-48 hr

Reading After 24-48 hrReading After 24-48 hr

Diagram of Procedure acc. to ISO/CD* draft 11290 and Diagram of Procedure acc. to ISO/CD* draft 11290 and AFNOR* Detection of Listeria monocytogenesAFNOR* Detection of Listeria monocytogenes

Grams staining

Catalase test

Motility test

Rhamnose:(+)

CAMP test

• Differentiation on PALCAM Agar Base is based on Esculin Differentiation on PALCAM Agar Base is based on Esculin hydrolysis and Mannitol fermentationhydrolysis and Mannitol fermentation

• Listeria Listeria spp. hydrolyzes esculin, which appears as spp. hydrolyzes esculin, which appears as blackening in the mediumblackening in the medium

• Mannitol and the pH indicator Phenol RedMannitol and the pH indicator Phenol Red

• Added to differentiate mannitol-fermenting strains of Added to differentiate mannitol-fermenting strains of possible contaminants, including enterococci and possible contaminants, including enterococci and staphylococci and appaer as yellow coloniesstaphylococci and appaer as yellow colonies

• Listeria Listeria spp do not ferment Mannitol and appaer as olive spp do not ferment Mannitol and appaer as olive green color colonies green color colonies

• Polymyixin B, Acriflavin, Ceftazidime, and Lithium Polymyixin B, Acriflavin, Ceftazidime, and Lithium Chloride are selective agents used to suppress Gram-Chloride are selective agents used to suppress Gram-negative and certain Gram-positive bacterianegative and certain Gram-positive bacteria

Palcam Agar Base - Enumeration Principle and Palcam Agar Base - Enumeration Principle and InterpretationInterpretation

++ =

Combines with Fe to give brown/black

color

Substrate- EsculinSubstrate- Esculin

Esculin + H2O = beta-D-glucose + 6,7-Esculin + H2O = beta-D-glucose + 6,7-dihyroxycoumarin dihyroxycoumarin

The rich basis of Chromocult Listeria Selective Agar is The rich basis of Chromocult Listeria Selective Agar is the addition of 5-bromo-4-chloro-3-indolyl-ß-D-the addition of 5-bromo-4-chloro-3-indolyl-ß-D-glucopyranoside which makes it possible to glucopyranoside which makes it possible to differentiate between ß -D-glucosidase positive and differentiate between ß -D-glucosidase positive and negative bacterianegative bacteria

Listeriae are ß -D-glucosidase-positive and grow on the Listeriae are ß -D-glucosidase-positive and grow on the medium in the form of blue-green coloniesmedium in the form of blue-green colonies

To detect To detect L. Monocytogenes L. Monocytogenes substrate L- ß -substrate L- ß -phosphatidylinositol is added to the medium phosphatidylinositol is added to the medium

L. monocytogenes L. monocytogenes has the enzyme phosphatidylinositol has the enzyme phosphatidylinositol phospholipase C (PI-PLCphospholipase C (PI-PLC) )

This phospholipase activity results in the formation of This phospholipase activity results in the formation of opaque haloes around opaque haloes around L. monocytogenes L. monocytogenes coloniescolonies

Chromocult Listeria Selective Agar Base - Enumeration Chromocult Listeria Selective Agar Base - Enumeration Principle and InterpretationPrinciple and Interpretation

Colony characteristics on Chromocult Listeria Selective Colony characteristics on Chromocult Listeria Selective Agar Base Agar Base

Enumeration of Enumeration of ListeriaListeria spp. spp. is based on the is based on the principle that the organism show tolerance to the principle that the organism show tolerance to the selective agent used in the isolation procedureselective agent used in the isolation procedure

Ability to hydrolyse esculin by the enzyme beta-Ability to hydrolyse esculin by the enzyme beta-glucosidaseglucosidase

Organism show weak beta-haemolysis on sheep Organism show weak beta-haemolysis on sheep blood agarblood agar

Ability to produce phosphatidylinositol-specific Ability to produce phosphatidylinositol-specific phospholipase-C (PI-PLC)phospholipase-C (PI-PLC)

Ability to ferment rhamnoseAbility to ferment rhamnose

So based on various formulation and combination So based on various formulation and combination different chromogenic and fluorogenic culture media different chromogenic and fluorogenic culture media have been developedhave been developed

Enumeration Enumeration PrinciplePrinciple

Screening of selective broths for enzymes activity by Screening of selective broths for enzymes activity by growing growing ListeriaListeria spp. and other potential contaminants spp. and other potential contaminants

Supplementation with Supplementation with Esculin and Ferric Ammonium CitrateEsculin and Ferric Ammonium Citrate resultsresults in blackening of broth or with in blackening of broth or with Chromogenic substratesChromogenic substrates results in blue and yellow colorationresults in blue and yellow coloration

II) Development of Selective Liquid Medium (SLM)II) Development of Selective Liquid Medium (SLM)

Sr No

Name of the Broth Marker Enzymes Color reaction at 37oC

1 Listeria Enrichment Broth (LEB)

ß-glucosidase

(PI-PLC)

α-d- mannosidase

Black

Blue

Yellow

2 Fraser Broth (FB)

3 University of Vermont Medium (UVM)

4 Brian Heart Infusion Broth (BHI)

Chromogenic substrateChromogenic substrate

For beta-glucosidase enzymeFor beta-glucosidase enzyme 5-Bromo-4 chloro3-indolyl-5-Bromo-4 chloro3-indolyl-ββ-D glucopyranoside (X-GLU)-D glucopyranoside (X-GLU)

PNPGPNPG

For For αα-mannosidase enzyme -mannosidase enzyme p-nitrophenyl ap-nitrophenyl alphalpha-mannoside-mannoside

For PI-PLC enzymeFor PI-PLC enzyme 5-Bromo-4-chloro-3-indoxyl myo-inositol-1 phosphate5-Bromo-4-chloro-3-indoxyl myo-inositol-1 phosphate

For beta-glucosidase enzyme For beta-glucosidase enzyme

4-methylumbelliferyl β-D glucoside4-methylumbelliferyl β-D glucoside

For alpha-D-mannosidase enzymeFor alpha-D-mannosidase enzyme

4-methylumbelliferyl-alpha-D-mannopyranoside4-methylumbelliferyl-alpha-D-mannopyranoside

For PI-PLC enzyme For PI-PLC enzyme

4-Methylumbelliferyl myo-inositol-1-phosphate4-Methylumbelliferyl myo-inositol-1-phosphate

Incubation at 37o C

Incubation at 37o C

Selective agent used in the isolation of Selective agent used in the isolation of Listeria Listeria spp.spp.

Antimicrobial AgentAntimicrobial Agent mg/Lmg/L Organism inhibited 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 ListeriaListeria spp. spp.Pure cells of Pure cells of ListeriaListeria spp. spp.

Spiked milk Spiked milk samples samples

Spiked milk Spiked milk samples samples

Natural milk Natural milk Natural milk Natural milk

Grown in SLM under optimised conditions Grown in SLM under optimised conditions

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