Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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previously unopened package of hot dogs manufac-

tured at the company’s plant in Zeeland, Michigan.

0071 In 1997, the plant was shut down for several days

for violating standard sanitation procedures, and in

July 1998 evidence of Listeria was found in the plant,

but not along the food production line. The plant was

supposedly aggressively sanitized and cleaned after

the July testing. Federal investigators suspect dust

kicked up during a summer maintenance project at

the meat-processing plant may have been the source

of the outbreak. The US Department of Agriculture,

which regulates the quality, sanitation, and safety of

meat products in the USA, found a dramatic increase

in the amount of Listeria levels on floors and equip-

ment in the plant in the 6 weeks after the construction

work, during which time the isolation rate for

‘Listeria-like indicator organisms’ on production

equipment rose from 25 to 92%. Maintenance on

the air-conditioning system performed in one of the

rooms at the plant could have caused widespread

contamination of meat products throughout the plant.

0072 In another outbreak involving processed meats, 29

illnesses caused by a strain of L. monocytogenes were

identified in 10 states between May and November

2000. The isolates from these cases were indistin-

guishable when subjected to genotyping by pulsed-

field gel electrophoresis and ribotyping. The outbreak

was linked to the consumption of contaminated

turkey and chicken deli meats produced at two estab-

lishments; one of these establishments produced

turkey meat for the second establishment.

Sporadic Cases

0073 Sporadic cases of listeriosis have arisen from the

consumption of meat and poultry products, dairy

products, vegetable products, and seafood. L. mono-

cytogenes counts of 3–5 10

were found on analyz-

ing samples of goats’ milk cheese implicated in one

case, and subsequent investigations suggested that

counts may have increased to these levels during stor-

age in the display cabinet of the retailer. This led one

researcher to propose that the increase in consump-

tion of relatively young soft cheese, coupled with

extended refrigeration of foods as family shopping is

carried out at weekly, or even monthly, intervals, may

be important factors contributing to listeriosis cases

reported in the UK.

See also: Hazard Analysis Critical Control Point; Lactic

Acid Bacteria; Listeria: Detection; Listeriosis

Further Reading

Farber JM and Peterkin PI (2000) Listeria monocytogenes.

In: Lund BM, Baird-Parker AC and Gould GW (eds) The

Microbiological Safety and Quality of Food, pp.

507–534. Gaithersburg, MD: Aspen.

Griffiths MW (1989) Listeria monocytogenes: its import-

ance in the dairy industry. Journal of the Science of Food

and Agriculture 47: 133–158.

Johnson JL, Doyle MP and Cassens RG (1990) Listeria

monocytogenes and other Listeria spp. in meat and

meat products. A review. Journal of Food Protection

53: 81–91.

Lovett J (1989) Listeria monocytogenes. In: Doyle MP (ed.)

Foodborne Bacterial Pathogens, pp. 283–310. New

York: Marcel Dekker.

Ryser ET and Marth EH (1999) Listeria, Listeriosis, and

Food Safety, 2nd edn. New York: Marcel Dekker.

Tompkin RB et al. (1999) Guidelines to prevent post-

processing contamination from Listeria monocytogenes.

Dairy, Food and Environmental Sanitarian 19(8):

551–562.

Various authors (1996) Special issue on Listeria monocyto-

genes. Food Control 7: 181–258.

Detection

M W Griffiths, Canadian Research Institute for Food

Safety, University of Guelph, Guelph, Ontario, Canada

Background

0001One of the primary goals of a food microbiologist is

to detect the presence of specific pathogens in foods in

the shortest possible time. This is not an easy task as

often, the organism of concern is present in low

numbers, and more often than not, it is found in

foods in the presence of large populations of other

bacteria. Detection of pathogens is primarily achieved

using a cultural approach involving essentially three

steps: (1) a nonselective and/or selective enrichment

procedure to increase the numbers of the target or-

ganism to detectable levels and provide an opportun-

ity for injured cells to recover, (2) a selective step

when the organism is presumptively identified and

(3) confirmation of the identity of the isolates

obtained by the first two phases.

Detection

0002The type of food being analyzed is an important factor

to consider when evaluating methods for recovery and

enumeration of specific bacteria, and L. monocyto-

genes is no exception. The physical nature of the food

may affect distribution of the inoculum on the medium

or mask color reactions important for colony differen-

tiation. More importantly, foods differ with respect to

LISTERIA

/Detection 3573

their indigenous microflora. Thus, a medium selective

for Listeria may successfully inhibit contaminants from

one type of foodstuff, but not from another. For this

reason, there is no one universally acceptable method

for the isolation of L. monocytogenes from foods, and

each regulatory jurisdiction has its own preferred

method.

Enrichment

0003 One of the most useful methods for enrichment of

L. monocytogenes involves incubation of samples at



C for up to 12 weeks in a nonselective medium.

Unfortunately, the length of this procedure precludes

its general use in the food industry.

0004 Experiments performed to optimize basal broth

medium for the growth of L. monocytogenes gave

rise to the formulation of an enrichment broth con-

taining proteose peptone (0.5%), phytone peptone

(0.5%), sodium chloride (0.5%), dextrose (0.1%),

yeast extract (0.1%), sodium pyruvate (0.1%), and

disodium phosphate (0.25%). Under optimum condi-

tions, the generation time of L. monocytogenes was at

least 6 min shorter in this broth than in tryptose

broth.

0005 A number of selective agents have been incorpor-

ated into enrichment broth. Most enrichment broths

for L. monocytogenes have contained nalidixic acid

to prevent growth of Gram-negative bacteria and

acriflavin to provide additional selectivity with regard

to Gram-positive organisms. A commonly used en-

richment broth, developed by the US Food and Drug

Administration, contains acriflavin, nalidixic acid,

and cycloheximide. Formulations based on these

inhibitors may not be entirely satisfactory due to the

growth of interfering species. Of special concern are

highly resistant strains of Enterococcus faecalis and

other enterococci. Reports of resistant strains of

Staphylococcus aureus and Kurthia spp. have also

appeared in the literature. An improved enrichment

formulation has been described by researchers at the

US Department of Agriculture (USDA). They claim

that buffered tryptose phosphate broth containing

acriflavin and claforan may be an effective enrich-

ment system, but still expressed some reserva-

tions about its ability to control the growth of all

enterococci.

0006 It has been claimed that two-stage enrichment pro-

cedures are more effective for amplifying the growth

of L. monocytogenes from food samples. In one such

procedure, UVM Listeria enrichment broth (contain-

ing acriflavin and nalidixic acid) was used initially.

After 24 h, samples were transferred to a modified

broth containing lithium chloride to inhibit entero-

coccal growth. The second enrichment broth also

contained esculin and ferric ammonium citrate so

that presumptive growth of L. monocytogenes could

be detected by the appearance of a black precipitate

due to the formation of iron phenolic compounds

resulting from esculin hydrolysis. No false negatives

were found when this technique was used on a variety

of dairy and environmental samples. Unfortunately, a

high rate of false positives was detected, which was

again primarily due to growth of enterococci. The

strongly buffered UVM broth has advantages when

isolating L. monocytogenes from soft cheese as it

combats the rapid drop in pH that results when

sampling this product.

0007Where there is likely to be heavy contamination

with interfering organisms, the more selective enrich-

ment broth, L-PALCAMY, may give better results.

However, the use of highly selective media may be

problematic as the high concentration of inhibitors in

the media can slow the growth of sublethally injured

or sensitive strains of Listeria. To overcome problems

associated with the recovery of sublethally injured

cells, a nonselective medium, such as buffered pep-

tone water, has been used as the primary enrichment

broth followed by secondary enrichment in a more

selective medium. A nonselective universal enrich-

ment broth has been described for the combined

enrichment of Listeria and Salmonella. It has also

been shown that strict anaerobic incubation aids in

the recovery of injured L. monocytogenes.

0008A two-stage enrichment of particular value for

meats involves primary enrichment for 24 h at 35



in proteose peptone–yeast extract broth followed by a

secondary enrichment under the same conditions in

the same broth, but this time modified to contain the

selective agents lithium chloride, glycine anhydride,

acriflavin, Tween 80, polymyxin B, ceftazidime,

phosphomycin, and nalidixic acid.

0009The resuscitation of L. monocytogenes depends on

the media, incubation conditions, and sample matrix.

For example, the recovery of L. monocytogenes from

cheese was affected by the method of homogenization

of the sample, the type of diluent used, and the tem-

perature of the diluent. The best results were obtained

when cheese samples were homogenized in a Waring

blender for 2 min and diluted in trisodium citrate (2%

w/v) pretempered to 40



C. However, due to safety

concerns, the authors recommended that blending be

replaced by stomaching for 3 min. When enrichment

was performed at 30



C, there was generally no ad-

vantage in prolonging the incubation time from 24 to

48 h. Optimum growth of L. monocytogenes during

enrichment is obtained if the initial pH of the broth

was between 6.8 and 7. Improved isolation of

L. monocytogenes was reported when the enrichment

was carried out under microaerophilic conditions

3574

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/Detection

(5% oxygen; 10% carbon dioxide; 85% nitrogen).

There was a decreased lag period noted for growth of

L. monocytogenes under microaerophilic conditions

with increased carbon dioxide concentrations. Dilu-

tion of the sample after enrichment with potassium

hydroxide is often recommended in protocols but is

of limited value, having no effect on the number of

recoveries or the background microflora. Enrichment

broths used for the detection of Listeria are shown in

Table 1.

Selection

0010 After amplification of the number of L. mono-

cytogenes cells by enrichment, a presumptive isol-

ation is carried out. This usually entails plating on

to selective or differential media. Several such plating

media for detection of L. monocytogenes have been

described (Table 2).

0011One formulation that has been used widely is

McBride’s Listeria agar. Phenylethanol, lithium chlor-

ide, and glycine serve as the selective agents, and

blood is incorporated into the medium to identify

hemolytic strains. Overgrowth of plates with resistant

staphylococci and streptoccoci can occur with this

medium. Modifications to this medium include the

incorporation of cycloheximide to prevent the growth

of yeasts and molds, and substitution of glycine

with glycine anhydride (a more effective selective

agent). Cephalosporin antibiotics such as moxalac-

tam have also been added to McBride’s agar to pro-

duce a highly selective medium, although periodic

problems due to growth of resistant isolates of enter-

ococci, Staphylococcus spp. and Kurthia spp. can

arise. Another cephalosporin, ceftazidime, may be

more effective than moxalactam. A medium contain-

ing ceftazidime and acriflavine as selective agents

inhibited all organisms, other than L. monocytogenes

tbl0001 Table 1 Composition of enrichment broths used for the detection of Listeria

Stage of enrichment/enrichment broth Acriflavine (mg l

1

) Nalidixic acid (mg l

1

)NaCl(gl

1

) Buffer Other additions

Primary enrichment

Buffered peptone water 0 0 5 Phosphate None

IDF standard 10 40 5 Phosphate Cycloheximide

Levinthal 15 40 5 None None

Lovett 15 40 5 Phosphate Cycloheximide

Modified Lovett K 10 40 5 Phosphate Cysteine; N

Modified Lovett MOPS 15 40 5 Phosphate/MOPS None

L-PALCAMY 5 0 5 None Ceftazidime; polymyxin;

LiCl; egg yolk

UVMI 12 20 20 Phosphate None

Universal 0 0 5 Phosphate MgSO

; pyruvate

Secondary enrichment

Fraser 25 20 20 Phosphate LiCl

Lovett 15 40 5 Phosphate Cycloheximide

UVM II 25 20 20 Phosphate None

Combined

Listeria repair broth 37 239 5 0 Phosphate/MOPS Cycloheximide; glucose;

yeast extract; MgSO

;

FeSO

; pyruvate

tbl0002 Table 2 Composition of Listeria selective agars

Agar Acriflavine (mg l

1

) Polymyxin (P)

or Colistin (C) (mgl

1

)

LiCl (gl

1

) Other additions (mg l

1

)

EHA 5 10 (P) 10 Ceftazidime (30)

LCA 0 0 5 Ceftazidime (50)

LPM 0 0 5 Moxolactam (20); glycine anhydride (10); phenylethanol (2500)

LSA 5 10 (P) 15 Ceftazidime (20); tellurite (200)

MC 0 10 (C) 12 Ceftazidime (20)

MVJ 0 0 5 Bacitracin (20); nalidixic acid (50); moxolactam (5); glycine (10);

tellurite (105)

Oxford 5 20 (C) 15 Fosfomycin (10); cefotetan (2); cycloheximide (400)

PALCAM 5 10 (P) 10 Ceftazidime (30)

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/Detection 3575

with the exception of a few enterococci, members of

the Enterobacteriaceae, yeasts, and molds.

0012 Differentiation of Listeria colonies on many media

is achieved by their characteristic blue to blue–gray

color when the plates are illuminated with obliquely

reflected light (Henry illumination). This technique

has been criticized because it is often difficult to

perform effectively, is subjective, and is not easily

quantifiable. Therefore, research has been concen-

trated on the development of media that rely on

other means of differentiation.

0013 A series of differential selective media have been

developed by researchers in The Netherlands. The

first, RAPAMY agar, used a Columbia blood agar

base with the addition of ferric ammonium citrate

and egg yolk emulsion. Selectivity was provided by

nalidixic acid, acriflavine, phenylethanol, and incu-

bation under microaerobic conditions. The presence

of esculin and ferric ammonium citrate together with

d-mannitol and Phenol Red results in presumptive

positive colonies that are dark gray with a black

center surrounded by a dense black halo (due to

esculin hydrolysis) against a cherry red background

(caused by the inability of Listeria to ferment manni-

tol). However, RAPAMY medium failed to isolate

Listeria from food in which Enterococcus spp.

exceeded Listeria by a factor of about 10

. A modifi-

cation of the medium to replace nalidixic acid with

lithium chloride to prevent growth of enterococci

gave encouraging results with a variety of foods,

including raw chicken, raw milk, raw vegetables

and raw milk soft cheeses. Enterococcus spp. and

Staphylococcus spp. that were able to grow on this

medium (ALPAMY) could be differentiated from

Listeria spp. on the basis of their colony morphology.

However, sublethally injured cells of L. mono-

cytogenes could not be recovered reliably on

ALPAMY. Further modification of the medium

resulted after addition of polymyxin B and ceftazi-

dime and supplementation of the Columbia agar base

with glucose. The modified medium (PALCAM agar)

recovered strains of L. monocytogenes and other Lis-

teria spp. quantitatively and suppressed growth of

most other bacteria commonly found in food. The

occasional Enterococcus or Staphylococcus isolates

that developed on the medium gave gray colonies

with a brown–green halo or yellow colonies with a

yellow halo.

0014 Another differential agar that has found wide-

spread use is Oxford agar. Again, Columbia agar

forms the basis of this medium, but the selective

agents used consist of lithium chloride, acriflavine,

colistin, cycloheximide, cefotetan, and fosfomycin.

Differentiation of Listeria spp. is obtained by the

incorporation of esculin and a ferric salt, resulting in

black zones around suspect colonies. Good inhibition

of interfering organisms, including enterococci, has

been reported using this medium.

0015Good results have been obtained for the isolation

of Listeria spp. from meats, poultry, and seafood

using a modified Vogel Johnson agar. The medium

includes glycine, elevated levels of lithium chloride,

nalidixic acid, bacitracin, and moxalactam as select-

ive agents. The medium also includes mannitol as a

differential agent and potassium tellurite as both a

differential and selective agent. On this medium, Lis-

teria spp. are entirely black on a red background,

whereas resistant strains of enterococci and staphylo-

cocci are surrounded by a yellow zone due to manni-

tol fermentation. The medium surrounding resistant

Kurthia strains turns maroon due to their strong

alkaline reaction.

0016Media for the presumptive identification of

L. monocytogenes based on the formation of the

enzyme phosphatidylinositol-specific phospholipase

C have been described. CHROMagar Listeria helps

to easily differentiate Listeria monocytogenes from

other Listeria directly at the isolation step. Listeria

monocytogenes colonies are blue and are surrounded

by a white halo due to a specific phospholipase activ-

ity. Formation of an opaque halo surrounding

L. monocytogenes colonies on Otaviani & Agosti

Listeria Agar (ALOA) permits identification of

L. monocytogenes even in mixed bacterial popula-

tions. When ALOA was compared with Oxford

medium for the detection of L. monocytogenes in a

variety of foods, including meat, meat products, sea

foods, egg products, dairy products and foods of plant

origin, the proportion positive for Listeria spp.

was 34.1% for ALOA compared with 30.4% for

Oxford medium. The proportion positive for L. mono-

cytogenes was 18.3% on ALOA and 3.6% on Oxford

medium. The chromogenic (5-bromo-4-chloro-3-

indoxyl-myo-inositol-1-phosphate) or fluorogenic

(4-methylumbelliferyl-myo-inositol-1-phosphate)

substrates have been added to media in order to iden-

tify the presence of the phospholipase.

0017The temperature at which the selective medium is

incubated may be important as the minimal inhibi-

tory concentration of Listeria spp. to four antibiotics

commonly used in selective plating media decreased

with increasing temperature. Strains tended to be

more susceptible to inhibition by the antibiotics at



C rather than at 30



0018It is unlikely that one medium will prove suitable

for the isolation of L. monocytogenes from all types

of food. In studies on direct plating of L. monocyto-

genes from foods, noncultured dairy products such as

pasteurized milk and chocolate icecream mix pre-

sented the fewest problems. However, Brie cheese

3576

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/Detection

and raw cabbage contained high levels of contamin-

ants that grew on isolation media. The main contam-

inants associated with Brie were molds, yeasts, and

Gram-positive cocci. High levels of Gram-positive,

rod-shaped contaminants were isolated with Listeria

spp. from cabbage, although Gram-negative rods

could often be detected on isolation media. Meats

also contained high populations of contaminating

microorganisms, principally Gram-positive cocci. Of

the food examined, raw oysters posed the biggest

problem because of the high numbers of indigenous

Gram-positive, rod-shaped bacteria present. In add-

ition, pseudomonads were often recovered from test

media plated with raw oysters.

Confirmation

0019 Confirmation of the identity of presumptive isolates

on selective agar is usually achieved by a study of

morphological and biochemical characteristics.

0020 L. monocytogenes is a Gram-positive, small, short

rod-shaped organism. These bacteria are flagellated

and exhibit a characteristic tumbling motility at



C. At higher temperatures (30–37



C), production

of flagella is suppressed with a concomitant loss in

motility. On solid medium, L. monocytogenes col-

onies are translucent but take on a blue–green color-

ation when viewed by transmitted light. On blood

agar, colonies produce a narrow zone of b-hemolysis.

0021 Listeriae are aerobic to microaerophilic, and

growth is enhanced in atmospheres with reduced

oxygen and 5–10% carbon dioxide. They possess a

heme-containing catalase and are oxidase-negative.

L. monocytogenes is positive for the Voges–Proskauer

and tellurite tests. The bacterium ferments glucose

with the production of mainly l(þ)-lactic acid, but

no gas. All members of the genus hydrolyze esculin

and maltose. Rhamnose and xylose reactions are

important in differentiating Listeria spp. The CAMP

test is also used to differentiate L. monocytogenes

from other members of the genus. Both L. seeligeri

and L. monocytogenes may not produce enough

hemolysin to be noticeably hemolytic. However,

when grown in an area of Staphylococcus aureus

growth, the hemolytic reaction of these Listeria spp.

is enhanced. The hemolysis is unreactive to metabol-

ites of Rhodococcus equi. Reactions used for differ-

entiation of Listeria spp. are summarized in Table 3.

0022Confirmation by biochemical tests can be made

simpler by the use of ready-to-use test strips and

automated identification systems such as the Vitek

Junior. The Vitek Gram-positive identification card

correctly identified 229 of 236 Listeria-positive isol-

ates, and 95% of these were confirmed within 6–8h.

Other miniaturized test kits for the identification of

L. monocytogenes based on phenotypical biochem-

ical reactions include an arylimidase test on paper

disks, API Listeria miniaturized test kit, Biolog

Micro-ID Listeria and Biolog Phenotype microarrays

for Listeria, and the Rosco system. When the Micro-

ID Listeria System, API Listeria, and the Rosco

system were compared with conventional identifica-

tion tests, all these rapid biochemical test kits gave

fast, reliable identification of Listeria species when

compared with classical techniques. However, the

API Listeria system identified the test strains without

a complementary CAMP test.

Detection in Processed Foods

0023The main difference between the detection of

L. monocytogenes in raw and processed foods is

that the latter may have been subjected to conditions

that sublethally stress the organisms of concern as

well as reducing their numbers. It is known that

L. monocytogenes can be sublethally stressed by

heat, acidulants, and, to a lesser extent, freezing.

Injured organisms need a period of recovery to repair

cellular damage. The resistance of sublethally injured

cells to selective agents used in their detection may be

substantially reduced. In general, the greater the

selectivity of the medium, the less likely it will be

that stressed cells will grow.

0024Injured Listeria cells are unable to grow in the

presence of 2% sodium chloride, and the proportion

of stressed cells may be determined by comparing

tbl0003 Table 3 Biochemical tests for the differentiation of Listeria spp

Test Listeriaspp.

monocytogenes innocua seeligeri ivanovii welshimeri

Hemolysis þþþ

CAMP – S. aureus þþ

CAMP – R. equi þ

Xylose þþþ

Rhamnose þ V

V

Variable reaction.

LISTERIA

/Detection 3577

counts on media with and without sodium chloride

addition. Using this technique, the effect of various

agents on the recovery of sublethally injured L. mono-

cytogenes has been determined. Selective agar used

for isolation of Listeria spp. generally did not allow

resuscitation of heat- or acid-injured cells. However,

freezing did not appreciably affect the ability of test

strains to grow on selective agar. The inability of a

medium to support colony formation by thermally

injured L. monocytogenes could be correlated with

the inclusion of phenylethanol, tellurite, polymyxin

B, sodium thiosulfate and oxgall, as well as sodium

chloride. The addition of Tween 80, bovine fetal

serum or egg yolk emulsion to modified Vogel–

Johnson medium increased the ability to detect heat

injured cells of the organism 100-fold without any

loss of selectivity of the medium.

0025 The universal response of cells undergoing injury

appears to be the accumulation of hydrogen peroxide,

and injured cells have an increased sensitivity to

the toxic effects of hydrogen peroxide. Removal of

hydrogen peroxide by the addition of catalase, or the

nonenzymatic peroxide decomposer sodium pyru-

vate, has a beneficial effect on the recovery of injured

cells of several microorganisms, including Staphylo-

coccus aureus and Salmonella typhimurium. Effective

repair of stressed L. monocytogenes cells on solid

medium containing catalase has been described.

Also, pyruvate addition to modified McBride agar

enhanced recovery of heat-injured L. monocytogenes,

but it was necessary to incubate plates at 22–25



C for

7 days in order to achieve beneficial effects. However,

the situation is confused as another study failed to

show enhanced recovery of L. monocytogenes Scott

A strain after addition of pyruvate to modified

McBride agar. The addition of reducing agents to

the medium has been shown to have a mixed effect

on the recovery of heat-injured cells of L. monocyto-

genes, but recovery is increased if cells are incubated

anaerobically. Enrichment in broth containing

oxygen scavengers, such as oxyrase, enabled recovery

of heat-injured listeriae within 6 h.

0026 When the effects of incubation temperature,

oxygen availability and supplementing the culture

medium with magnesium chloride, magnesium sul-

phate, d-glucose, l-cysteine, catalase, or lithium

chloride on the recovery of salt- or acid-damaged

Listeria monocytogenes serovar 1/2c was studied on

a solid repair medium, results showed that conditions

promoting resuscitation of acid- or salt-injured cells

are stress-specific, and differ in part from those

described for heat-stressed Listeria.

0027 It is strongly recommended that a nonselective

enrichment is included when processed foods are

analyzed for L. monocytogenes. An incubation time

of 6–9 h at temperatures in the range of 20–40



Cis

required for complete recovery of injured cells. The

temperature optimum appears to be 25



C. The

organism does not appear to be able to repair at low

temperatures. Prevention of large pH changes during

the enrichment by buffering of the medium also

promotes recovery of heat-stressed cells.

Detection in Suspected Food Poisoning

0028Confirmation of the involvement of L. monocytogenes

in a case of illness suspected to be of food origin relies

on isolation of identical strains of the organism from

the patient and the suspected food. In practice, this is

not feasible in the majority of cases, and epidemi-

ological case-control studies have proved crucial in

implicating a specific food product in an outbreak.

The epidemiological surveillance of listeriosis has

been hampered by the lack of adequate typing

schemes. A serotyping system is available, but is of

limited value as a high proportion of strains causing

disease belong to only a small number of serovars. Of

all L. monocytogenes strains isolated in Britain from

human cases of listeriosis, 91% comprised only three

serovars: 4b (59%), 1/2a (18%), and 1/2b (14%). A

phage-typing scheme is also used, but a high propor-

tion of strains (about 36% in Britain) are not typable

by this system. An international multicenter typing

study of L. monocytogenes was initiated by the

WHO (Food Safety Unit, Geneva) in order to evaluate

the usefulness of various phenotypic and genotypic

typing methods for L. monocytogenes and to select

and standardize the most appropriate methods. Sev-

eral genotyping methods were compared, including

pulsed field gel electrophoresis (PFGE) coupled

with the use of the restriction enzymes, ApaI and

SmaI, random amplification of polymorphic DNA

(RAPD), high frequency restriction endonuclease an-

alysis (REA), and restriction-fragment length poly-

morphism (RFLP). In general, this study reconfirmed

that PFGE is a very accurate and reproducible method

for fine structure comparison and molecular typing of

L. monocytogenes. An automated genotyping system,

the RiboPrinter, has proved useful for the analysis of

L. monocytogenes and was used successfully to deter-

mine the source of the outbreak linked to hot dogs

produced at the Bil Mar plant of Sara Lee. L. mono-

cytogenes has been classified into three genetic lin-

eages using ribotyping and PCR-RFLP typing of

hlyA and octA genes. A higher proportion of human

isolates (69.1%) than industrial isolates (36.8%) were

classified as lineage I, which contains human sporadic

isolates and all epidemic isolates. All other industrial

isolates (63.2%) were classified as lineage II, which

contains only human sporadic isolates.

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Other Methods of Detection

0029 As well as the classical cultural approach to the de-

tection of Listeria spp., more sophisticated techniques

are available commercially. These have advantages in

their ease of use and in rapidity.

Impedance Monitoring

0030 When microorganisms grow in a medium, large-

molecular-weight substrates are catabolized to more

highly charged, low-molecular-weight end products.

These modify the electrical properties of the medium,

and the changes can be detected as changes in con-

ductance or capacitance. A selective medium has been

described that contains acriflavin, ceftazidime, and

nalidixic acid for use with impedance monitors such

as Bactometer, Malthus, and RABIT. The medium

also contains esculin and a ferric salt. The presence

of Listeria spp. is confirmed by detection of growth

and black coloration of the medium. In addition,

growth of Listeria spp. gives a characteristic response

curve when the capacitance signal is monitored. The

medium gave good results with fermented dairy

products, but there appears to be a high rate of false

positives when soft cheeses are analyzed. A medium

(LED medium) based on the selective agents used in

Oxford agar has also been described for the electrical

detection of Listeria spp. The medium relied upon the

ability of Listeria spp. to induce a greater than 30%

change in capacitance within 30 h. When compared

with a traditional Fraser broth enrichment, the

method gave far fewer false-positive results when

used to analyze environmental swabs and products,

such as meat, cream cakes, cheeses, and milks.

0031 An indirect conductimetric assay that detects

carbon dioxide evolved during growth of micro-

organisms is available. Such methods allow the in-

corporation of high concentrations of salts, such as

lithium chloride, into media to aid selective growth of

Listeria spp.

Immunological Methods

0032 Monoclonal antibodies specific for Listeria spp. have

been produced in a number of laboratories. There are

several commercially available immunoassays for

Listeria and L. monocytogenes (Table 4). A commer-

cially available system applicable to foods involves

the use of two monoclonal antibodies with an

enzyme-linked immunosorbent assay (ELISA).

Following enrichment in broth for 40–48 h at 30



the ELISA procedure is performed. One antibody is

used for antigen capture, and one for enzyme conju-

gation with horseradish peroxidase. For the ELISA to

be effective, the enrichment broth must contain at

least 10

Listeria cells per milliliter. The method was

able to identify all milk samples containing L. mono-

cytogenes. However, the detection rate with raw

vegetable samples was only 68%. This was probably

due to inadequacies in the enrichment procedure

caused by low levels of Listeria spp. in the presence

of mixed microflora. The method lacks species speci-

ficity within the genus Listeria. A monoclonal anti-

body specific for strains of L. monocytogenes, L.

innocua,andL. welshimeri has been isolated.

0033A direct immunofluorescence test has been used to

detect successfully L. monocytogenes in naturally

contaminated soft cheese samples when numbers

exceeded 10

per gram. The test could be carried

out in 2 h. An immunomagnetic capture assay has

been developed that is capable of detecting less than

2 10

Listeria cells per milliliter in enriched foods.

0034An automated instrument, Vidas, has been de-

veloped consisting of a solid phase receptacle (SPR)

and pipetting device. The SPR is coated with anti-

bodies against the target antigen, and the necessary

reagents to perform an enzyme-linked fluorescent

assay are contained in a ready-to-use reagent strip.

The fluorescence measured is directly proportional to

the quantity of antigen present in the sample. Two

tests are available: the VIDAS LIS method detects

Listeria spp., and the VIDAS LMO method is claimed

to be specific for L. monocytogenes. The VIDAS LIS

method was compared with two conventional culture

methods for detection of Listeria spp. in naturally

contaminated or inoculated ice cream, cheese, green

beans, fish, roast beef, and turkey mince. The agree-

ment between the VIDAS LIS and culture methods for

all samples tested was 86%. The VIDAS LIS method

has been approved by AOAC International for detec-

tion of Listeria in foods. When used to detect

L. monocytogenes in cheese, the Vidas assay gave

positive results in 97.5% of all contaminated cheeses

after 48 h of enrichment. The detection limit was

6.3 10

to 2.0 10

cfu ml

1

0035A comparison of commercially available immuno-

logical kits for detection of Listeria spp. (Listeria

Rapid Test Clearview, Pathalert Listeria, VIDAS Lis-

teria LIS, VIP Listeria) and Listeria monocytogenes

(Pathalert Listeria monocytogenes, VIDAS Listeria

monocytogenes LMO) in food showed that there

were no significant differences between the tests,

but all methods had difficulties with the detection of

L. monocytogenes.

Gene Probes and Related Techniques

0036A nonisotopic, colorimetric nucleic acid hybridiza-

tion assay based on 16S rRNA sequences for the

detection of Listeria spp. in dairy and environmental

samples has been described. After a two-stage

LISTERIA

/Detection 3579

enrichment procedure, the hybridization assay is per-

formed, permitting detection of the organism in

about 60 h. The procedure was able to identify 98%

of Listeria-positive milk samples but only 45% of

positive raw vegetable samples. The latter results

were thought to be due to inadequate growth of the

organism in the enrichment broth used.

0037 A DNA probe specific for b-hemolytic L. mono-

cytogenes has been applied to colonies obtained on

selective media inoculated with food samples. The

method was rapid and accurate for detecting the

pathogen in raw milk, semisoft cheese, and ricotta

cheese. The method was quantitative, provided that

the contamination level in the food was greater than

10 cells per gram. Enrichment was necessary if

numbers of L. monocytogenes were below this level.

A probe, specific for L. monocytogenes has been

obtained from two DNA fragments encoding part of

the listeriolysin O molecule.

0038Two DNA hybridization assays for L. monocyto-

genes have been commercialized (Table 5). One, the

Gene-Trak Listeria monocytogenes Assay, has been

compared with the IDF Standard 143A:1995 using

107 cheese, fish, and meat samples naturally contam-

inated with Listeria. The Gene-Trak assay gave a

significantly higher number of positive results. Exam-

ination of a further 75 cheese and meat samples with

both methods and, additionally, with three immuno-

logical tests (Listeria Rapid Test Clearview, VIDAS

Listeria LIS, and a noncommercial ELISA) again

confirmed that the Gene-Trak assay gave a greater

number of positive samples than all the other

methods. The differences, however, were not signi-

ficant. Another commercially available gene probe

tbl0004 Table 4 Commercial immunoassays for Listeria spp. and L. monocytogenes

Company Test name Specificity Time to perform Principle

BioMe

rieux, Inc. Vidas Lis Listeria spp. 45 min (excluding enrichment) Automated enzyme-linked

fluorescent assay

Vidas LMO L. monocytogenes 45 min (excluding enrichment)

TECRA International

Pty Ltd

Listeria Via Listeria spp. 48 h (including enrichment) Visual immunoassay

Listeria Unique Listeria spp. 32 h (including enrichment) Antibody-coated dipstick

BioControl VIP for Listeria Listeria spp. Single-use, visual

immunoprecipitate

assay

Assurance Listeria L. monocytogenes and

related species

30 min (excluding enrichment) Polyclonal enzyme

immunoassay,

microplate with

breakaway wells

Neogen Reveal for Listeria Five of six Listeria

spp. including

L. monocytogenes

43 h (including enrichment) Antibody against specific

flagellar antigen; ELISA

sandwich immunoassay

combined with

chromatography in a

single-use device

Hardy Diagnostics Listeria Rapid Test kit

Vicam Listertest Listeria spp. 24 h Immunomagnetic beads

coupled with selective

media and confirmation

with membrane lift

immunoassay

Diffchamb Transia Plate Listeria Listeria spp. 24–48 h Based on a sandwich-type,

immunoenzymatic

reaction using a mixture

of antibodies

Transia Plate

Listeria monocytogenes

Listeria monocytogenes Based on three-step,

sandwich-type,

immunoenzymatic

reaction; solid support of

the reaction is a

microtiter plate coated

with antibodies specific

for the extracellular P60

protein excreted by

Listeria monocytogenes

3580

LISTERIA

/Detection

assay for L. monocytogenes (AccuProbe) correctly

identified samples containing the organism. However,

it has been determined that some enrichment media

(University of Vermont broth and Fraser broth) inter-

fered with the AccuProbe test, due to prevention of

cell lysis by the high salt concentration in these media.

The detection limit of the AccuProbe test was also

affected by enrichment and selective broths.

0039 The polymerase chain reaction (PCR), using

thermostable DNA polymerase, permits the rapid

amplification of specific DNA sequences by a factor

of up to 10

. The portion of DNA amplified is deter-

mined by oligonucleotide sequences (primers).

Primers have been synthesized that result in PCR

products specific for L. monocytogenes. PCR proto-

cols based on amplification of the listeriolysin O gene

(hlyA), the internalin gene (inlA), a gene encoding a

metalloproteinase (mpl), and the gene encoding an

invasive-associated protein (iap) are among those

that have been developed. The products can be

detected by agarose gel electrophoresis, but more

recently, real-time PCR methods have been developed

that allow the detection of amplified products using

fluorescence.

0040 To overcome problems due to inhibition of the

PCR by components of food, several methods have

been investigated such as the use of filters to entrap

and lyse bacterial cells, the use of immunomagnetic

separation, and magnetic capture hybridization.

0041Commercial kits to detect L. monocytogenes based

on PCR have become available (Table 5). The Bax

system has been semiautomated and has been shown

to be an effective method of screening for L. mono-

cytogenes in smoked fish and many different types of

fresh produce.

0042Undoubtedly, with the further development of real-

time PCR methods and the advent of DNA array tech-

nology, molecular methods will be increasingly used for

routine testing of foods for L. monocytogenes.

See also: Food Poisoning: Classification; Tracing Origins

and Testing; Immunoassays: Radioimmunoassay and

Enzyme Immunoassay; Listeria: Properties and

Occurrence; Listeriosis; Salmonella: Salmonellosis;

Staphylococcus: Food Poisoning