
listeriosis cases have been shown to be caused by
serotypes 1/2a, 1/2b, and 4b. Further analyses has
revealed that serotype 4b is prevalent in many Euro-
pean countries. In North America, serotypes 1/2a
(Canada) and 4b (USA) are most often recovered in
cases of listeriosis. Again, the reason for the differ-
ences observed in serotype and geographical distribu-
tion is unclear. Equally, it is not known at present why
only three serotypes cause most of the illness.
Food and Clinical Diagnosis
0007 The food microbiologist has the task of analyzing a
food product for the presence of L. monocytogenes.
They must be able to achieve this goal as rapidly as
possible, as the food implicated may have already
been consumed prior to the identification protocol
being completed. Because listeriae are found through-
out nature and in a wide variety of foods, there is
no universal protocol at the moment. The majority of
identification methods for the detection and enumer-
ation of L. monocytogenes are cultural in nature.
Other methods include the use of DNA probes, amp-
lification-based methods, antibody-based technolo-
gies, and other rapid methods.
0008 In Canada, the Bureau of Microbial Hazards
(part of the Health Products and Food Branch)
uses a detection method developed for all food
and environmental samples (Figure 1). Presumptive
colonies are then subjected to biochemical testing,
using the criteria listed in Table 2 to differentiate
L. monocytogenes from other species. Enumeration
would involve macerating the sample with peptone
water and plating on to any of two LPM, Oxford,
MOX, or Palcam agar plates, followed by incubation
at 30
C (LPM) or 35
C (Oxford, MOX, Palcam).
0009The cultural method utilizes selective agents with
the hope of allowing cells of L. monocytogenes to
grow and competing flora to be inhibited. LEB, also
known as University of Vermont broth, contains
nalidixic acid and acriflavin as selective agents.
LPM medium contains the Gram-positive and
Gram-negative inhibitory compounds, lithium chlor-
ide, phenylethanol, and moxalactam. Fraser broth
contains lithium chloride, nalidixic acid, and acrifla-
vin. The addition of esculin and ferric ammonium
citrate causes a darkening of the broth or agar in the
presence of Listeria species (Listeria species have the
ability to hydrolyze esculin, and hydrolysis of esculin
turns the medium black, denoting a presumptive posi-
tive test). Oxford agar contains lithium chloride,
cycloheximide, colistin, acriflavin, fosfomycin, and
cefotetan as selective agents, in addition to esculin
and ferric ammonium citrate. Palcam agar and broth
contain, in addition to esculin and ferric ammonium
citrate, lithium chloride, polymixin-B-sulfate, ceftazi-
dime, and acriflavin. Interestingly, it would appear
that Palcam is preferred in Europe, while LPM,
Oxford, and modified Oxford are used predomin-
antly in North America. Many laboratories across
the world are currently working on novel formula-
tions that one day may be superior to currently used
media.
0010Other methods have been reported in the literature
for the detection and enumeration of L. monocyto-
genes in foods. The first report of a monoclonal anti-
body specific for the genus Listeria was reported in
1987. Since then, antibody-based detection methods
have been described. These are based on the enzyme-
linked immunosorbent assay. Many companies offer
commercially available kits that use the antibody–
antigen–antibody sandwich technique as the basis
for detection. Antibodies have also been used in con-
junction with magnetic beads. The beads are covered
with Listeria-specific antibodies and mixed with the
sample to be tested. A magnetic field is applied to
separate the magnetic beads on which the Listeria
cells are bound. After a washing step, other methods
(for example, cultural methods) may be applied.
Monoclonal antibodies have been used to serotype
the genus Listeria, supplemented by hemolysis of
blood and in vivo mouse assays (Table 3).
0011There are many protocols described that have, as
their basis, hybridization of genetic material (DNA).
Many probes have been described and used in colony
hybridization methods, as have polymerase chain
Blend or stomach sample in LEB broth and
incubate at 30 C for 48 hours.
Confirmation tests on presumptive isolates.
Tests include Gram-stain, catalase,
hemolysis, motility,
mannitol/rhamnose/xylose acid production,
CAMP test, serology, biochemical or rapid
identification kits.
Streak positive modified Fraser broths onto
selective agar plates. Reincubate negative
Fraser broths for an additional 24 hours.
Incubate the agar plates for 24 to 48
hours.
At 24 and 48 hours, transfer 0.1 ml of LEB
into modified Fraser broth. Incubate 24 to
48 hours at 35 C. (Optional: streak LEB
onto plates).
fig0001 Figure 1 Flow chart for the isolation of L. monocytogenes from
environmental and food samples.
LISTERIA
/Listeriosis 3583