1. Conventional Method for the
Detection of Listeria spp. From
Dairy Products

2. 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

3. 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

4. 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

6. 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

8. 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

9. 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

10. 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

11. 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

12. General scheme for the isolation of Listeria spp. from
food products

13. 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

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

15. Grams staining

16. Catalase test

17. Motility test

18. Rhamnose:(+)

19. CAMP test

20. 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

21. Substrate- Esculin
+
=
+
Combines with Fe to give
brown/black color
Esculin
+
H2O
= beta-D-glucose
dihyroxycoumarin
+
6,7-

22. 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

23. Colony characteristics on Chromocult Listeria
Selective Agar Base

24. 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

25. 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

26. 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

27. Incubation at 37 o C
Incubation at 37 o C

28. 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

29. 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

30. Thank you