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Journal of Food Protection, Vol. 65, No. 11, 2002, Pages 1811–1829
Copyright Q, International Association for Food Protection
Review
Listeria monocytogenes Virulence and Pathogenicity, a Food
Safety Perspective
SOPHIA KATHARIOU*
Food Science Department and Program in Genomic Sciences, North Carolina State University, 339 Schaub Hall,
Raleigh, North Carolina 27695, USA
ABSTRACT
Several virulence factors of Listeria monocytogenes have been identiŽ ed and extensively characterized at the molecular
and cell biologic levels, including the hemolysin (listeriolysin O), two distinct phospholipases, a protein (ActA), several
internalins, and others. Their study has yielded an impressive amount of information on the mechanisms employed by this
facultative intracellular pathogen to interact with mammalian host cells, escape the host cell’s killing mechanisms, and spread
from one infected cell to others. In addition, several molecular subtyping tools have been developed to facilitate the detection
of different strain types and lineages of the pathogen, including those implicated in common-source outbreaks of the disease.
Despite these spectacular gains in knowledge, the virulence of L. monocytogenes as a foodborne pathogen remains poorly
understood. The available pathogenesis and subtyping data generally fail to provide adequate insight about the virulence of
Ž eld isolates and the likelihood that a given strain will cause illness. Possible mechanisms for the apparent prevalence of three
serotypes (1/2a, 1/2b, and 4b) in human foodborne illness remain unidentiŽ ed. The propensity of certain strain lineages
(epidemic clones) to be implicated in common-source outbreaks and the prevalence of serotype 4b among epidemic-associated
strains also remain poorly understood. This review Ž rst discusses current progress in understanding the general features of
virulence and pathogenesis of L. monocytogenes. Emphasis is then placed on areas of special relevance to the organism’s
involvement in human foodborne illness, including (i) the relative prevalence of different serotypes and serotype-speciŽc
features and genetic markers; (ii) the ability of the organism to respond to environmental stresses of relevance to the food
industry (cold, salt, iron depletion, and acid); (iii) the speciŽ c features of the major known epidemic-associated lineages; and
(iv) the possible reservoirs of the organism in animals and the environment and the pronounced impact of environmental
contamination in the food processing facilities. Finally, a discussion is provided on the perceived areas of special need for
future research of relevance to food safety, including (i) theoretical modeling studies of niche complexity and contamination
in the food processing facilities; (ii) strain databases for comprehensive molecular typing; and (iii) contributions from genomic
and proteomic tools, including DNA microarrays for genotyping and expression signatures. Virulence-related genomic and
proteomic signatures are expected to emerge from analysis of the genomes at the global level, with the support of adequate
epidemiologic data and access to relevant strains.
Listeria monocytogenes is the only species in the genus
Listeria that is of concern for human health. This facultative
intracellular, gram-positive bacterium is capable of causing
serious invasive illness (listeriosis) in both humans and animals (62, 97, 179, 180). The transmission of this pathogen
by contaminated food was Ž rst conclusively demonstrated
by epidemiologic and laboratory investigations in 1983
(176) and has been shown to cause both sporadic cases and
outbreaks of listeriosis. Certain segments of the population,
including the elderly, neonates, pregnant women, human
immunodeŽ ciency virus–infected individuals, and individuals undergoing immunosuppressive therapy, are at increased risk of infection. In nature, the primary habitat of
Listeria appears to be soil and decaying vegetation. Unlike
most human pathogens, L. monocytogenes can grow at refrigeration temperatures (78). The ubiquitous distribution of
* Author for correspondence. Tel: 919-513-2075; Fax: 919-515-7124;
E-mail: skathar@unity.ncsu.edu.
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MS 01-481: Received 18 December 2001/Accepted 23 March 2002
this bacterium in the environment, its ability to grow in the
cold, and its pathogenic potential make this pathogen of
particular concern for the safety of refrigerated and readyto-eat (RTE) foods consumed without reheating, cooking,
or both. Several outbreaks of listeriosis have been traced to
contaminated cold-stored RTE foods, including dairy, vegetable, and meat products (179). Earlier reviews have addressed in detail the bacteriology and human epidemiology
of L. monocytogenes (62, 91, 97, 179).
As a facultative intracellular pathogen, L. monocytogenes can survive and grow in mammalian cells, including
phagocytes. Protective immunity is cell mediated, as Ž rst
shown by Mackaness (119), with many investigations having addressed the components of the listerial cell-mediated
immune response (for reviews, see (35, 100)). These fundamental attributes of the pathogen, along with the fact that
it can be grown easily in the laboratory, have rendered it
an effective model system for the study of bacterial pathogenesis, intracellular survival, cell biology of host-patho-
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KATHARIOU
1.0. VIRULENCE AND PATHOGENESIS
1.1. Overview of virulence determinants and interactions of L. monocytogenes with host cells: 1.1.a. The
listeriolysin O region. The genes for several key virulence
determinants of L. monocytogenes, including the hemolysin
(listeriolysin O), two phospholipases, and a protein (ActA)
essential for intracellular motility of the pathogen, are located in one well-deŽ ned gene cluster in the chromosome
of the bacterium. Upon infection of host cells, the bacteria
are internalized in a vacuole. Expression of listeriolysin O
promotes escape from the vacuole into the cytoplasm,
where the microbe replicates. The intracytoplasmic bacteria
use the actin of the host cell, in conjunction with their ActA
protein, to promote their motility intracellularly, their location in protruding pseudopods, and the engulfment of the
pseudopods by adjacent host cells. After their uptake by
adjacent cells, the bacteria escape the now double-membrane–bound vacuole by means of listeriolysin O and the
phospholipases, and the cycle repeats. The process, exhibited by L. monocytogenes, Shigella  exneri, and certain
Rickettsia spp., is as striking at the electron microscopic
and cell biologic level today as it was when Ž rst described
(190).
The production of listeriolysin O in the host cell is
under stringent regulatory control. In the murine model, a
genetically constructed variant with a single amino acid
change that rendered the protein more stable in the cytoplasm of the infected cell was found to have a decrease in
virulence of more than 3 logs (49). Clearly, this is a virulence factor that is expressed in the vacuole but not in the
cytosol, thus preventing killing of the host cell and allowing
the host cell cytoplasm to serve as a safe haven for bacterial
survival and replication. Similar regulatory controls may
well exist for other virulence factors.
Expression of the virulence genes mentioned above requires the transcriptional activator, PrfA, encoded by a gene
in the same genomic cluster. PrfA-mediated regulation has
been extensively studied (see reviews in (56, 89, 97)).
1.1.b. Determinants involved in cell invasion, intracellular motility, and cell-to-cell spread. In the mouse
model, L. monocytogenes has been shown to invade enterocytes or M cells in Peyer’s patches (117, 166). The bacterium replicates in enterocytes and in highly permissive
mononuclear cells in the Peyer’s patches, in a process that
is still being studied (165), and disseminates to the primary
target organs (liver and spleen). Most bacteria that reach
the liver are killed by resident macrophages (114, 119). The
few that survive are likely to be cleared by the host’s normal immune system. However, any survivors can go on to
infect hepatocytes and can eventually cause systemic infection and invasion of the secondary target organs (central
nervous system, placenta, and fetus).
In model cell culture systems, adherence and invasion
of host cells require several determinants. Most intensively
studied are the surface proteins internalin A (InlA) and internalin B (InlB), which are differentially required for infection of different cell types and recognize different receptors on the host cells (27, 127). The association of L.
monocytogenes with these receptors leads to phosphorylation of various host cell proteins and a complex signal
transduction cascade that results in pathogen-mediated internalization of the bacteria. As will be discussed later (see
section 1.2.1.a), the receptor for InlA (E-cadherin) is not
functional in murine cells. However, transgenic mice that
harbor a functional E-cadherin are much more sensitive to
wild-type L. monocytogenes than to isogenic InlA-deŽ cient
mutants, indicating an important role of InlA in virulence
in vivo (109). Several other invasion determinants of L.
monocytogenes have been identiŽ ed, suggesting that the
cell adherence and invasion process is complex and multifactorial (for reviews, see (40, 56, 76, 89, 97, 103)). The
cell adherence-invasion process of Listeria is still being actively studied (27, 130).
Actin-based intracellular motility of L. monocytogenes
is crucial to cellular pathogenesis and requires ActA, encoded by a gene in the listeriolysin O gene region. ActA is
both essential and sufŽ cient for Listeria actin-based motility, and actA mutants are avirulent in mice. Mechanisms of
ActA action, along with other components of the pathogen’s intracellular motility and cell-to-cell spread, have
been extensively studied (reviewed in (67, 89)).
Genetic studies continue to identify genes essential for
the virulence of L. monocytogenes (e.g., (12, 41, 68, 109)).
Most studies, however, have utilized strains EGD and
10403S (both serotype 1/2a) or strain LO28 (serotype 1/2c)
and may not effectively address virulence attributes speciŽ c
to other serotypes that are of clinical importance, such as
1/2b and 4b. Strains of serotypes 1/2a and 1/2c belong to
one of two major genetic divisions in L. monocytogenes,
found to be quite distinct from the division that includes
serotypes 1/2b and 4b (21, 22, 31, 156); see also section
2.1.3). Although the key virulence factors known to date
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gen interactions, and cell-mediated immunity. Intracellular
replication of the pathogen appears to be intimately connected with virulence as well as with induction of protective immunity, as intracellular growth and processing of
selected antigens is required for effective immune responses
(19, 35).
Since the middle 1980s, an impressive body of knowledge has accumulated concerning the molecular biology of
virulence determinants of this microorganism and the cell
biology of its interactions with host cell receptors, the cytoskeleton, and signal transduction pathways. This work has
been reviewed extensively (for recent reviews, see (40, 56,
76, 89, 103)). Hence, these studies will not be reviewed
here. Furthermore, although these studies have contributed
greatly to our understanding of listerial (and general) cellular pathogenesis, they have not adequately addressed or
elucidated the special and numerous virulence-related concerns that the pathogen raises for food safety. This review
details and discusses our current understanding of some key
issues in this regard, with special attention drawn to the
different serotypes and clonal lineages found in foods and
implicated in foodborne listeriosis; the lineages responsible
for epidemics; and the avenues for effective detection of
pathogenic strains in foods and in the community.
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L. MONOCYTOGENES VIRULENCE AND PATHOGENICITY
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for routine studies and surveys of virulence, these results
highlight the continuing need for alternative models, especially in regard to infections via the oral or i.g. route.
In earlier studies, i.v. and i.g. infections were used to
determine the impact of bacterial growth temperature on
virulence. Interestingly, low-temperature growth enhanced
virulence in i.v. but not in i.g. infections (44, 186), suggesting that caution should be exerted in interpreting virulence data obtained following different routes of infection.
1.2. Laboratory determinations of virulence—animal and cell culture models: 1.2.1. Murine models. Murine models have been used extensively, with virulence
quantitated in terms of the persistence of the bacteria in the
spleen or liver following intravenous (i.v.) infections (87,
100). A widely used model involves determinations of 50%
lethal dose for immunocompromised mice (185).
1.2.1.b. Brain infection models. Efforts have been
made to model central nervous system invasion in foodborne infection, with maternal encephalitis detected in the
pregnant mouse model, depending on the timing of infection in regard to gestation (128). Repeated oral dosing (5
3 109 over 7 to 10 days) resulted in severe brain lesions
(with histopathology similar to that seen in human encephalitis) in 25% of the mice (6). Neurovirulence of the pathogen after direct brain inoculation also has been studied
(177). Recent studies have pursued the identiŽ cation of
genes essential for bacterial multiplication in the brain of
mice following i.v. infections (see also section 2.4), using
signature-tagged mutagenesis, a method that allows the
identiŽ cation of mutants that are attenuated in virulence and
thus fail to be recovered from the target organ (brain). Interestingly, a large number of mutants were found to harbor
lesions in the gene gtcA, which varies noticeably in sequence between serotypes 4b and 1/2a (12). In serotype 4b,
gtcA is essential for serotype-speciŽ c glycosylation of wallassociated teichoic acid (164).
1.2.1.a. Oral and intragastric infections. To better
simulate aspects of foodborne infection, oral and intragastric (i.g.) infection models have been used (14, 158, 175).
Certain strains that had similar virulence in i.v. infections
were poorly infective in terms of spleen colonization following i.g. inoculation, suggesting that i.g. infection may
be a more discriminatory model for detecting potential virulence differences (14). Mice immunosuppressed by cyclosporin were more sensitive to i.g. infection than untreated
animals and were used in experiments to demonstrate the
impact of intestinal bacterial  ora on the outcome of oral
infection by L. monocytogenes (150). Corticosteroid treatment in an oral infection model also has been used, resulting in prolonged infections (161). Using oral infections in
pregnant mouse models, Lammerding et al. (104) and Menudier et al. (128) were able to monitor maternal and fetal
infection. Strains were found to differ in terms of their ability to cause fetal infection following oral inoculation during
pregnancy (104).
The experimental study of oral infections in the murine
model has been hampered by the apparent innate resistance
of mice following oral or i.g. routes of infection, a fact that
requires the use of high inocula (commonly 109 to 1010
bacteria) and that likely accounts for the commonly observed lack of reproducibility of the virulence estimates.
Unlike humans, mice (and rats) lack a functional receptor
for InlA (E-cadherin), and this may compromise efforts to
evaluate enterocyte invasion following oral and i.g. infections using murine and rat models. The inability of murine
and rat E-cadherin to bind InlA was localized in a speciŽ c
amino acid substitution. Guinea pigs, in contrast, were
found to harbor a human-type E-cadherin (108). These data
may explain previous results, which suggested that InlA
was not essential for virulence in the murine model. Recently, however, it was shown that transgenic mice that harbored the human form of E-cadherin in their enterocytes
were much more susceptible to oral infections by wild-type
L. monocytogenes (serotype 1/2a) than by a mutant that
lacked the gene for InlA, suggesting that InlA may be a
key virulence factor for animals expressing a functional Ecadherin receptor, including humans (109). Although transgenic mice would represent a cumbersome animal model
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(listeriolysin O, phospholipases, ActA, internalins A and B,
and others) are present in all serotypes, their regulation of
expression may differ among serotypes. Furthermore,
strains of serotypes 1/2b and 4b may have additional virulence determinants, which will not become identiŽ ed until
and unless strains of these serotypes become included in
genetic studies of virulence. The identiŽ cation of potentially unique virulence factors of these strains may be aided
by comparative genomic analyses (see also section 4.4)
1.2.2. Other animal models. As mentioned earlier,
mice (and rats) lack a functional receptor for the protein
InlA, which is required for listerial invasion of human intestinal epithelial cells in culture. The guinea pig, which
has a functional receptor, has been proposed as a better
model in this regard (108, 109). The chick embryo model
(146) also has been used to compare virulence of different
isolates (13, 141, 147) on the basis of 50% lethal dose or
percentage mortality in infected embryos. The examination
of a panel of strains with the chick embryo model and the
murine model (i.v. infection) suggested agreement between
the two (146, 147).
1.2.3. Cell culture models. To bypass the practical and
ethical problems posed by animal models, as well as to
better address speciŽ c components of the pathogenesis process, several teams have developed cell culture models.
These models can determine the ability of L. monocytogenes to attach to cells grown in culture, invade, and disseminate from one infected cell to another. Using the human
intestinal epithelial cell Caco-2, Pine et al. (157) showed
that L. monocytogenes was the only Listeria spp. virulent
in this assay and demonstrated strain-speciŽ c differences.
Although pronounced differences were reproducibly found
among strains, no obvious correlations with serotype or
source (clinical versus food or sporadic versus epidemic)
were identiŽ ed. Van Langendonck et al. (200) also used
Caco-2 cells to differentiate among strains that had differences in virulence in the immunodeŽ cient mouse model.
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KATHARIOU
Cell plaque-forming assays have been used to screen food
and clinical isolates of L. monocytogenes (144). In addition,
cell culture assays determining the cytotoxicity of L. monocytogenes have been described (20).
1.3.1. Cold and plant-derived molecules repress virulence gene expression. Under laboratory conditions, the
production of listeriolysin O and several other virulence
factors is repressed at temperatures below ca. 258C (99,
113), suggesting that virulence genes may be repressed
when the bacteria are growing in cold-stored foods or in
cold environmental niches in nature. Interestingly, virulence
gene expression is also repressed by plant-derived molecules such as cellobiose and the phenolic compound arbutin, both of which may be encountered by the organism in
soil, in nonpathogenic situations (28, 152, 153). Thus, L.
monocytogenes appears to be able to detect signals speciŽ c
to at least two fundamentally distinct habitats (warm-blooded animals versus soil and vegetation) and to express or
repress speciŽ c sets of genes accordingly.
The repression of virulence gene expression at 48C
does not affect proper expression of the genes following
infection (in response to the body temperature of the host
and other signals in vivo). Interestingly, strains may differ
in their ability to resume growth at 378C following cold
storage, with clinical isolates resuming growth more readily
than strains derived from raw meat (13), suggesting that the
physiology of the transition from low temperature to human
body temperature may be relevant to the infectious process.
1.3.2. Response systems to iron depletion. Recent
biochemical and molecular studies have produced novel information concerning the possible impact of environmental
stresses such as iron deprivation and acid stress on virulence. L. monocytogenes does not produce siderophores to
sequester iron from its growth medium, but it may be able
to use exogenous siderophores produced by other microorganisms in its vicinity (181a). Interestingly, esculetin, the
hydrolysis product of the plant glycoside esculin, neutralizes the effect of iron-chelating agents and increases the
virulence of the bacteria in the murine model (42). The
growth of L. monocytogenes in iron-depleted media also
requires a general stress response protein (ClpC), which, if
mutated, renders the organism avirulent in the murine model (170, 171). In addition, these mutants are sensitive to
heat, salt, and oxidative stresses in synthetic media (but not
in complex media such as brain heart infusion) (171). Another stress protein (ClpE), required for survival during
prolonged exposure to high temperature, may also contribute to virulence in the murine model (137). In earlier studies, heat shock and other stresses appeared to induce virulence-related proteins (183).
1.3.3. Acid tolerance and acid stress. Acid tolerance
of L. monocytogenes is of interest, since this pathogen is
exposed to low pH at several stages during infection, including the acidic environment of the stomach and the phagosomal environment. Following phagocytosis of L. monocytogenes by macrophages, the phagosome is rapidly acidiŽ ed, with this acidiŽ cation being a prerequisite for the escape of the bacteria into the cytoplasm, where they replicate
(50). Interestingly, L. monocytogenes is noticeably less tolerant to low pH than Escherichia coli O157:H7 or S.  exneri (46). This is in agreement with epidemiological evidence that links antacids and reduced stomach acidity with
susceptibility to listeric infection (75, 86) and with similar
results obtained from an oral infection model (175). Nonetheless, L. monocytogenes does exhibit a degree of acid
tolerance, which is dependent on the medium and microbial
growth phase (46, 48), with acid-adapted bacteria having
enhanced survival in acidiŽ ed dairy products and other low
pH foods (70).
Using a biochemical approach that employed two-dimensional gels and mass spectrometry, Phan-Thanh and
Mahouin (155) showed that acid stress (pH 3.5) and acid
adaptation (pH 5.5) resulted in the induction of the expression of 47 and 37 proteins, respectively, including 23 proteins in common. Although the speciŽ c functions of these
proteins and their possible impact on virulence remain to
be determined, several Ž ndings suggest that the acid resistance of L. monocytogenes is important in pathogenesis. An
acid-sensitive mutant of L. monocytogenes was found to be
reduced in virulence in the mouse model, whereas an acidtolerant mutant had increased virulence (41, 122, 148). The
latter observation is of interest, as it suggests that certain
acidic conditions may select for variants of the microbe
with enhanced virulence.
Interestingly, no effects on virulence were seen when
a protein (sB ) required for the transcription of several general stress genes of L. monocytogenes was mutated, even
though the mutants had impaired acid resistance (208). Certain components of the acid and general stress resistance
system may be redundant so that their inactivation does not
impair virulence noticeably, at least in the model systems
that were employed.
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1.3. Environmental stresses (iron deprivation, acidity, and osmolarity) and virulence. In foods and the environment, as well as in vivo during infection, L. monocytogenes is exposed to numerous stress signals, which may
strongly in uence its pathogenicity. Stresses because of refrigeration, dehydration, freezing and freeze-thawing, heat,
acid, and salt, as well as exposure to disinfectants and other
antimicrobial substances, are of special relevance to the
physiological status and virulence of this pathogen in foods.
Work in this area has involved model systems with bacteria
grown in liquid batch cultures (planktonic cells) and may
not adequately re ect the state of the microbe in the food
processing facilities, the foods themselves, and the tissues
of the host, where the bacteria are attached to surfaces,
often as components of bioŽ lms that may involve other
microorganisms. Recent reports have highlighted the food
safety relevance of bioŽ lm formation in the contamination
of processing plants by the pathogen (191).
J. Food Prot., Vol. 65, No. 11
1.3.4. The impact of cold and salt tolerance on virulence. The extent to which cold and salt stress signals may
affect virulence gene expression has not been determined.
Improved, simpliŽ ed assays to detect cold-osmotic adaptation (25) and virulence gene expression (69, 110) will be
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L. MONOCYTOGENES VIRULENCE AND PATHOGENICITY
useful in this regard. Although genes involved in salt and
cold tolerance have been identiŽ ed (15, 16, 101, 182, 211),
the impact of these genetic determinants on virulence is not
clear. A recent study showed that mutants deŽ cient in oligopeptide transport were not only cold sensitive but were
also unable to survive intracellularly in macrophages, even
though overall virulence was not affected (26), suggesting
that interference with metabolic processes essential for lowtemperature growth may have pleiotropic effects on certain
aspects of cellular pathogenesis.
2.0. EPIDEMIOLOGICAL ASPECTS
2.1.2. The use of molecular subtyping for surveillance of human listeriosis. Pulsed-Ž eld gel electrophoresis
protocols and data sharing via the Internet have been utilized to implement PulseNet, a national network coordinated by the Centers for Disease Control and Prevention and
currently involving 46 state laboratories, 2 local public
health laboratories, and Food and Drug Administration as
well as U.S. Department of Agriculture food safety laboratories (187). Canadian laboratories now participate in
PulseNet, and it is likely that additional international laboratories will join the network. The epidemiological and
public health impact of such a standardized molecular subtyping network cannot be overemphasized. Commonsource outbreaks can be detected while still in early stages,
and clusters involving diverse strains can also be readily
identiŽ ed. PulseNet is expected to continuously augment
our knowledge on trends in the incidence of the pathogen,
the contribution of different strain types to human illness,
and the possible emergence of different lineages implicated
in sporadic or epidemic listeriosis.
2.1.3. Serotype-associated clonal structure of the
species. Over 10 years ago, multilocus enzyme electrophoresis (MEE) showed that serotypes 1/2a, 1/2c, 3a, and 3c
belonged to a major genetic division that was distinct from
the division that includes serotypes 1/2b, 3b, and 4b (21,
22, 156). However, both divisions contain serotypes prevalent in human illness (1/2a, 1/2b, and 3b). These typing
results showed that the genetic structure of the species is
clonal, i.e., consists of genetically distinct lineages (156).
The observed clonal structure in L. monocytogenes suggests
that horizontal gene  ow between the different serotypeassociated clonal lineages is limited.
It is intriguing that the H ( agellin) antigens of L. monocytogenes used in the serotyping scheme devised by Seeliger and Hoehne (181) correlate so precisely with the genetic partitioning of the species. The genetic clusters identiŽ ed by these early MEE investigations have been conŽ rmed by numerous alternative typing schemes, including
ribotyping (77, 209), pulsed-Ž eld gel electrophoresis (31,
135), and, more recently, computerized analyses of random
ampliŽ ed polymorphic DNA (123) and ampliŽ ed fragment
length polymorphisms (1, 5). In addition, sequencing or
restriction fragment length polymorphism analysis of numerous L. monocytogenes genes (including genes encoding
listeriolysin O and other virulence genes,  agellin, and p60,
as well as a genomic region essential for low-temperature
growth) could differentiate strains of serotypes 1/2a, 1/2c,
3a, and 3c from those of serotypes 1/2b, 3b, and 4b (167,
202, 203, 212). Although clonal lineages are common in
bacterial pathogens (e.g., (199)), the clonal partitioning of
the species along serotypic groups is not routinely seen in
other pathogens that possess multiple serotypes (2). These
Ž ndings may have substantial implications for food safety,
as they suggest that strains of certain serotype-associated
lineages may pose much higher risks to human health than
others.
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2.1. Serotype-associated species partitioning in L.
monocytogenes: 2.1.1. Incidence of different serotypes in
human illness. Although 12 serotypes can cause disease,
at least 95% of L. monocytogenes strains isolated from human listeriosis cases (both outbreak and sporadic) are of
three serotypes: 1/2a, 1/2b, and 4b (168a, 179, 189, 200a).
The prevalence of these serotypes in illness has been documented in numerous surveys from different countries (9,
47, 106, 124, 168a). It is not clear whether the observed
serotype distribution re ects potential differences in human
virulence and other, as-yet unidentiŽ ed, attributes of the organism’s ecology and physiology (including survival and
growth in foods) that make strains of these serotypes more
likely to colonize food processing environments and contaminate cold-stored RTE foods at infectious levels. In one
study, correlations were identiŽ ed between certain serotypes and clinical presentation (e.g., prevalence of serotype
4b in pregnancy-associated cases) (124), suggesting variation in human virulence among serotypes.
1815
2.1.4. Genetic structure of L. monocytogenes: additional insights. The typing results have provided some additional interesting information about the genetic structure
of L. monocytogenes, particularly as noted in the four paragraphs that follow.
(i) Genetic diversity differs among serotypes. Certain
serotypes are much more diverse genetically than others,
with serotype 1/2a being the most diverse. In the study by
Bibb et al. (22), serotype 1/2a exhibited 30 distinct MEE
types (in contrast to 10 and 11 for serotypes 4b and 1/2b,
respectively). This diversity of serotype 1/2a has been conŽ rmed by other typing investigations (1, 32, 93, 135). The
relatively low genetic diversity of serotype 4b also has been
noted (77) and may suggest that this serotype emerged relatively recently, as has been discussed in regard to certain
other animal pathogens (198). Furthermore, all screened
strains of serotype 1/2c were genetically indistinguishable
with the typing tools that were used (1, 36, 138), suggesting
that this clonal lineage may be very young, may be under
strong selective pressures, or both. This Ž nding is intriguing, considering that serotype 1/2c is often prevalent in
foods and food processing environments (see section 3.2).
(ii) Food-derived strains have pronounced genetic diversity. Food-derived strains were more genetically diverse
than clinical strains (21), suggesting that variable niches
and conditions select for diverse genotypes in foods and
food processing environments and that only certain foodderived lineages may become implicated in human illness.
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KATHARIOU
2.2. Serotype 4b L. monocytogenes—epidemiology
and ecology: 2.2.a. Clinical prevalence. Several surveys
of clinical listeriosis, in different countries, indicate that the
overall incidence of serotype 4b is high, ranging from 50
to 70% (9, 106, 120, 124). Strains of serotype 4b account
for a substantial fraction of sporadic infections and numerous common-source outbreaks of listeriosis (91, 97), and
the presence of bacteria of this serotype in an RTE food
appears to increase the food’s risk of implication in listeriosis (159). Strains of serotype 4b tend to be overrepresented in perinatal listeriosis (124, 140), suggesting that
they may have special virulence attributes for pregnancy
and breach of the blood-placenta barrier.
2.2.b. Prevalence in foods. It must be emphasized that
current data on the prevalence of serotype 4b (and other
serotypes) in RTE foods in the United States are not commonly available. However, earlier studies surveying various
foods suggested that serotype 4b was not the leading serotype among food isolates (79, 84). The discrepancy between food incidence and prevalence in illness may suggest
that strains of serotype 4b are more virulent to humans than
other serotypes, although other possibilities, including
transmission by routes other than contaminated foods, cannot be excluded.
The reasons for the apparent scarcity of serotype 4b
among most food-derived strains are unclear. It is possible
that other serotypes are better adapted to food and food
processing environments. Indeed, strains of serotype 1/2c
were found to adhere to stainless steel surfaces more efŽ ciently than those of serotype 4b (145). There may also be
niche speciŽ city for serotype 4b, which would allow it to
establish itself and become prevalent only in certain microhabitats in food processing facilities. Such aspects of the
ecology of this serotype as well as other serotypes of L.
monocytogenes are currently poorly understood and in need
of study. It is also possible that serotype 4b may be more
sensitive to selective enrichment protocols than other serotypes, with its presence in foods and the food processing
environments being underestimated. The fact that different
strains of L. monocytogenes are differentially sensitive to
isolation protocols has been documented (115, 172). Alternative isolation protocols and DNA-based methods will be
needed to better monitor and detect serotype 4b bacteria in
food and food processing environments.
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The development of tools to differentiate between clinical
and food isolates is discussed in a separate section (3.4).
(iii) Common strains infect both humans and animals.
The same genotypes are commonly found in human and
animal isolates (156), with the exception of a lineage that
is associated primarily with animal listeriosis but rarely
found among human clinical isolates (209).
(iv) Strains of serotypes other than 1/2a and 1/2c are
relatively understudied. Most genetic and immunologic
studies of L. monocytogenes have involved strains of serotype 1/2a (strains 10403S, EGD, NCTC 7973, and Mack)
or 1/2c (strain LO28), with these strains representing only
one of the two major genomic divisions of the pathogen.
Strain EGD (serotype 1/2a), isolated from an epidemic of
listeriosis in a rabbit laboratory colony in 1924 (136) and
used for numerous immunologic, bacteriologic, and genetic
studies thereafter, was chosen for the L. monocytogenes genome sequencing project (74). It is imperative that strains
representing the other major genomic division be actively
studied, both genetically and in terms of their physiology,
virulence, and ecology. This is dictated not only by the
documented genetic distance between the two genomic divisions, but also by the fact that strains of serotypes 1/2b
and 4b are of substantial epidemiologic and clinical importance. From this perspective, the choice of a serotype 4b
epidemic-associated strain for the genome sequencing by
the U.S. Department of Agriculture-The Institute for Genomic Research (www.tigr.org) is especially opportune.
J. Food Prot., Vol. 65, No. 11
2.2.c. Environmental prevalence. Two separate studies from Russia and Italy, respectively, suggest that 4b is
the prevalent serotype in sewage (52, 120). This is significant, considering that L. monocytogenes has been repeatedly shown to persist in treated sewage and sewage sludge
(3, 4, 72, 116, 118, 204). In the study by De Luca et al.
(52), L. monocytogenes (with 4b as the prevalent serotype)
was primarily recovered from activated sludge but not from
anaerobic digesters, suggesting that the pathogen was resistant to biological oxidation. The reported incidence of L.
monocytogenes serotype 4b in sewage is surprising and
may re ect human carriage of this serotype, subclinical infections, or resistance of the bacteria to environmental conditions in activated sludge (such as oxidation, UV light, and
phages). In fact, serotype 4b was found to be predominant
among strains from asymptomatic human carriers (118). No
recent studies of L. monocytogenes in sewage in the United
States have been reported, and human carriage studies in
this country are similarly lacking. Important epidemiological and public health functions will be served by such studies and by the typing of prevalent L. monocytogenes lineages recovered from sewage.
2.3. Epidemiology of serotypes 1/2a and 1/2b. Even
though these two serotypes account for a substantial portion
of sporadic listeriosis and occasional outbreaks, virtually
nothing is known about their potentially unique features of
pathogenesis, transmission, and ecology. In a study by
McLauchlin (124), serotype 1/2b was primarily associated
with nonpregnant individuals with severe underlying illness
and was found in 10% of all cases. In a survey in Los
Angeles County, California, the incidence of serotype 1/2b
was 31% among listeriosis cases (excluding those associated with known foodborne outbreaks) but was noticeably
higher (65%) among human immunodeŽ ciency virus–infected patients, suggesting a possible association of infection by this serotype with special dietary factors or sexual
practices (60). Alternatively, severe types of immunosuppression (e.g., following human immunodeŽ ciency virus infection) may allow infection by serotype 1/2b strains that
may be relatively noninfectious for individuals in other risk
categories, such as pregnancy.
These Ž ndings suggest that the relative incidence of
serotype 1/2b is likely to be variable in different surveys,
depending on the prevalence of individuals with severe im-
J. Food Prot., Vol. 65, No. 11
L. MONOCYTOGENES VIRULENCE AND PATHOGENICITY
munocompromising conditions. As discussed earlier, at the
genomic level, serotype 1/2b strains appear to be closely
aligned with serotype 4b and quite distinct from serotype
1/2a. Nonetheless, the two serotype groups (1/2b and 4b)
clearly represent distinct clonal lineages that differ in terms
of their surface antigenic composition (see section 2.4 below) and, possibly, their virulence and ecological niche in
foods and the environment.
2.4.2. Serotypes other than 4b. Both of the other predominant clinical serotypes, 1/2a and 1/2b, have teichoic
acid that contains N-acetylglucosamine and rhamnose substituents. Thus, this teichoic acid composition (common in
all serogroup 1/2 strains) is distinct from that of serotype
4b. Rhamnose and N-acetylglucosamine substituents on the
teichoic acid of serotype 1/2 L. monocytogenes are essential
for phage adsorption (192, 205). Since rhamnose is a teichoic acid component characteristic of serotype 1/2, the
gene or genes for its incorporation in the teichoic acid may
also be unique. Currently, serotype 1/2a or 1/2b mutants
that speciŽ cally lack rhamnose are not available, and the
effects of these substituents on virulence and interactions
with host cells are not known. However, rhamnose may be
involved in the attachment of the Ž rst component of complement C1q to the cell walls of the L. monocytogenes of
serotype 1/2 (7).
The study of gene cassettes essential for teichoic acid
glycosylation in serotype 4b (111, 164) led to the identiŽ cation of genomically equivalent genes in other serotypes.
Interestingly, the serotype 4b gtcA gene has a divergent
counterpart in serotype 1/2a and all other screened serotypes and, in addition, is preceded by a gene (mtrA) that
lacks any homologous counterpart in serotype 4b and that
may, on the basis of DNA and deduced protein sequence
analysis, encode a glycosylation enzyme (105).
A recent study utilized signature-tagged mutagenesis to
identify mutants of strain EGD (serotype 1/2a) that were
unable to multiply in the brains of mice. Interestingly, a
large number of mutations were localized in the gene gtcA,
which varies noticeably in sequence between serotypes 4b
and 1/2a (12, 105). It is currently not known whether gtcA
has similar virulence functions in serotype 4b. The possible
virulence role of the newly identiŽ ed mtrA gene, which is
absent from serotype 4b but present in all other major serotypes, remains to be determined.
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2.4. Serotype-speciŽ c genes and surface antigens:
2.4.1. Serotype 4b. Serotype designations in Listeria follow an alphanumeric system based on  agellin antigens (the
letter portion of the designation) and somatic antigenic determinants (the numeric portion of the designation). The
latter correspond primarily to sugar substituents of the anionic cell wall polymer, teichoic acid, which is covalently
bound to the peptidoglycan. In serotype 4b, teichoic acid
has a unique composition, with both glucose and galactose
substituents attached to N-acetylglucosamine in the teichoic
acid chains (66, 96, 195). Recently, two genomic regions
that are required for the incorporation of galactose, glucose,
or both in the teichoic acid of serotype 4b bacteria were
identiŽ ed. Mutations in these regions abolish reactivity with
a panel of monoclonal antibodies speciŽ c for serotype 4b
(and the antigenically similar serotypes 4d and 4e) (98, 112,
164). In both cases, a serotype-speciŽ c gene cassette that
is  anked by genes conserved among different serotypes
was identiŽ ed (111, 164). Interestingly, in strains of other
serotypes, the serotype-speciŽ c space is occupied by unrelated sequences, which are also unique to serotypes other
than serotype 4 (105, 111). The availability of serotypespeciŽ c sequences will allow the implementation of DNAbased assays (e.g., by polymerase chain reaction) to more
accurately survey foods and food processing facilities for
the presence of L. monocytogenes serotype 4b. The unique
teichoic acid composition of serotype 4b may have important ecologic and pathogenesis functions. The presence of
galactose appears essential for the attachment of serotype
4b–speciŽ c phages and for the invasion of mammalian cells
in culture (163). The sugar substituents on teichoic acid are
immunodominant determinants (98, 196) and may thus be
important in pathogen-host interactions and in the generation of subsequent protective immunity. Interestingly, although these surface antigens are very stable in laboratory
cultures and in sporadic clinical strains, population-level
surveys of different outbreaks identiŽ ed several strains that
lacked galactose in their teichoic acid (38). It is possible
that such strains have been selected in the course of infection as an evasion strategy toward the host immune system
(see also section 2.7).
1817
2.5. Epidemic-associated L. monocytogenes. Even
though most incidences of human listeriosis are sporadic,
it is foodborne outbreaks that have earned special notoriety
for L. monocytogenes. Implicated foods have included milk
and dairy products, cold-smoked salmon and other Ž sh and
seafood products, vegetables, coleslaw, and RTE meat
products. In certain cases, the foods have been epidemiologically associated but not conŽ rmed bacteriologically,
whereas in many of the well-publicized outbreaks, the implication of a speciŽ c contaminated food was based on both
epidemiologic analysis and bacteriologic conŽ rmation (59,
62, 90, 132, 134).
Bacteriologic characterization of the strains from outbreaks has failed to identify discrete determinants common
to all. In Europe and North America, most publicized outbreaks in the past 20 years have involved serotype 4b.
Strains of other serotypes, however, are not exempt from
the potential to cause outbreaks, as will be discussed below.
Several efforts using cell culture and animal models have
failed to identify differences in virulence between epidemic-associated strains and most other strains of the same serotype (33, 157, 158).
2.5.1. Epidemic clone I. In spite of their apparent bacteriologic conformity, many epidemic-associated serotype
4b strains appear to be genetically distinct from other
strains of the same serotype. Results from several subtyping
schemes suggest that the strains implicated in several geographically and temporally distinct outbreaks are closely
related (32, 33, 90, 156), even though each outbreak population is genetically distinct (32, 206). This group of
strains includes those implicated in outbreaks in Nova Sco-
1818
KATHARIOU
2.5.2. Epidemic clone II. From 1998 to 1999, a new
genotype of L. monocytogenes serotype 4b was implicated
in a multistate outbreak of listeriosis in the United States
that involved contaminated hot dogs. Strains from this outbreak had unique ribotype and pulsed-Ž eld gel electrophoresis patterns not commonly encountered in previous surveys (132, 133). Thus, the strains from this outbreak appear
to represent a novel epidemic-associated lineage, designated
epidemic clone II.
The involvement of an ‘‘unusual’’ serotype 4 strain in
an outbreak has been described at least once previously. In
1987, 23 cases of listeriosis in the United Kingdom were
attributed to a strain of an unusual serotypic designation,
4b(X) (125). Although an implicated food was not identiŽ ed, the bacteriologic and genotypic characterization of the
strains suggested that they represented a unique epidemic
clone rarely seen before or afterward.
2.5.3. Epidemic clone III. The most recent multistate
outbreak of listeriosis in the United States involved contaminated turkey deli meat products and resulted in several cases and a massive product recall (134). Unlike most other
outbreaks, the implicated strain was serotype 1/2a. An especially interesting Ž nding was that the outbreak strain was
the same genotype as a strain that was implicated in a human listeriosis case associated with the consumption of
contaminated turkey franks in 1988 (131). The products
implicated in the 2000 multistate outbreak were from the
same food processing facility as the earlier isolate, suggesting that this strain had persisted there over several years
without detectable genotypic changes (188). Genetic features unique to and characteristic of epidemic clone III remain to be identiŽ ed.
2.6. Epidemic strains: ampliŽ cation and reservoirs.
The periodic (and often repetitive) involvement of the same
epidemic clone in different outbreaks suggests that the implicated strains have a reservoir during the often lengthy
intervals between outbreaks. The widespread incidence and
epidemiology of ECI suggests that this clonal group may
be a ubiquitous environmental lineage, which can become
ampliŽ ed in animals and humans. No information currently
exists as to the speciŽ c environmental niche or niches (e.g.,
soil, silage, sewage, and others) for this group. ECI has
been frequently isolated from food animals (24, 142), suggesting that farm animals and the animal-associated environment, including animal feed, may serve as both temporary reservoirs and a means for ampliŽ cation. Human
carriers, or subclinically infected individuals, may also
serve as reservoirs for this clone. Routine surveys of food
have only sporadically yielded ECI strains, suggesting that
this lineage, which is clearly virulent to humans and farm
animals, does not commonly contaminate the food processing environment. The sources and possible reservoirs of
epidemic clones II and III, which caused multistate outbreaks in the United States in 1998 to 1999 and 2000, respectively, remain unidentiŽ ed. As mentioned earlier, epidemic clone III strains appeared to have persisted in the
processing plant for more than a decade.
Current data on possible reservoirs and ampliŽ cation
hosts for this pathogen need to be viewed in the context of
the organism’s potential to contaminate food and the food
processing environment. However, substantial gaps remain
in our knowledge of the transmission of foodborne listeriosis. The earlier conclusion of the World Health Organization working committee on foodborne listeriosis that L.
monocytogenes should be viewed as an environmental contaminant (8) may still be largely true. This view, however,
may need to be updated to take into account other sources
that also may be important, including the handling and
cross-contamination of food in food service settings such
as restaurants, delicatessens, and salad bars and by the consumer in the home. More survey work is needed to clarify
the contribution of these segments of the farm-to-fork continuum to the burden of foodborne listeriosis.
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tia (1981), Massachusetts (1983), California (Jalisco
cheese, 1985), Switzerland (1983 to 1987), Denmark (1985
to 1987), and France (1992).
Several of these outbreak strains (Nova Scotia, California, and Switzerland, but not Massachusetts) share a
unique restriction fragment length polymorphism in a genomic region essential for low-temperature (48C) growth of
L. monocytogenes (212). In addition, these same strains also
appear to methylate cytosines at GATC sites in their DNA,
which renders the DNA resistant to digestion by the restriction enzyme Sau3AI (213). Recently, several DNA sequences unique to these strains were identiŽ ed, although
the functional roles of the genes were not determined (85).
These and other Ž ndings suggest that these strains belong
to a distinct lineage, designated epidemic clone I (ECI). The
reported genetic similarity between the strains in ECI and
the strain implicated in the French pork-tongue-in-aspic
outbreak of 1992 (90) suggests that this strain also belongs
to ECI. Thus, ECI appears to be a cosmopolitan epidemic
clonal lineage. In this context, the choice of an ECI strain
(Jalisco outbreak) for the serotype 4b genome sequence determination by the U.S. Department of Agriculture-Agricultural Research Service seems especially appropriate.
J. Food Prot., Vol. 65, No. 11
2.7. In situ evolution in common-source outbreaks
of listeriosis. Outbreaks are population-level events. Even
when one common source is involved, outbreaks involve
multiple inocula, multiple infection events, and multiple
hosts, each with unique immune system idiosyncrasies. It
would therefore be expected that the pathogen-host interaction might have different dynamics in each case within
an outbreak and that the pathogen might have the opportunity to adapt independently in each host. Such an adaptation will yield genetic variants, which may or may not be
easily detectable genotypically and phenotypically. The
identiŽ cation of such variants by necessity requires the
study of multiple strains from a speciŽ c outbreak population.
Variation within the outbreak population has indeed
been observed. Twenty-seven percent of the patient-derived
strains from the Nova Scotia outbreak lacked galactose substituents in the teichoic acid of the cell wall. Such strains
were negative with serotype 4b–speciŽ c monoclonal anti-
J. Food Prot., Vol. 65, No. 11
L. MONOCYTOGENES VIRULENCE AND PATHOGENICITY
2.8. Involvement of other L. monocytogenes serotypes (1/2a, 1/2b, and 3a) in foodborne listeriosis outbreaks. Even though serotype 4b has been implicated in
most of the well-publicized outbreaks, several outbreaks involving other serotypes of L. monocytogenes strains have
been reported. In fact, the Ž rst recorded outbreak of listeriosis, which took place in Halle (formerly East Germany)
in 1945, involved serotype 1/2a (151). More recent outbreaks in Western Australia (1978 to 1980 and 1990 to
1991) and Auckland, New Zealand (1992), have involved
serotypes 1/2a and 1/2b (29, 193). A recent outbreak in
Finland was attributed to the contamination of butter by a
strain of serotype 3a (116a) and, as mentioned earlier, turkey deli meat products contaminated by bacteria of serotype
1/2a were implicated in a recent multistate outbreak in the
United States (134). Furthermore, in the last decade, strains
of serotypes 1/2a and 1/2b have been involved in epidemics
of febrile gastroenteritis in the United States and Europe
(45, 129, 162, 174). An outbreak of febrile gastroenteritis
has also been caused by serotype 4b (10).
Most listeriosis cases are sporadic and frequently involve serotypes 1/2a and 1/2b (179). At least some of the
apparently sporadic cases, however, may in reality represent
clusters of unrecognized outbreaks, especially if they occur
over a long period. In fact, high-resolution strain typing has
conŽ rmed this hypothesis in Austria (5), with this situation
likely to occur elsewhere as well. Similar recognition of
‘‘hidden’’ outbreaks will become more frequent in the United States as typing data (pulsed-Ž eld gel electrophoresis)
from ‘‘sporadic’’ cases continue to enter the PulseNet database. Because many sporadic cases are serotypes 1/2a and
1/2b, it is likely that at least several of these outbreaks,
which would otherwise be unrecognized, will prove to be
caused by these serotypes.
2.9. Role of dairy and meat animals in the transmission of listeriosis. In its early era, listeriosis was often
described as a zoonosis—an illness transmitted from animals to humans (180)—but zoonotic transmission of invasive illness has been seldom documented, and this view is
not widely held currently. Some animal listeriosis cases are
caused by Listeria ivanovii, well known as an animal pathogen but extremely rare in human infections, and by serotype 4a and 4c strains of L. monocytogenes, which are
found more frequently associated with animal than human
listeriosis and which constitute a unique lineage (209).
However, most strains from animal listeriosis are not readily distinguishable genetically from those implicated in human illness (156, 209). Hence, the question arises as to
whether animals may transmit such strains to humans.
The transmission from farm animals to humans may
occur under certain circumstances. In a 23-year (1972 to
1994) survey in Denmark, Jensen et al. (92) found that
herds harbored L. monocytogenes at a low but constant level (0.2 to 4.2% of herds). During the same period, 79% of
the isolates from bovine mastitis and 48% of the human
clinical isolates had overlapping ribotypes, suggesting that
milk and other dairy products from mastitic cows accounted
for at least some human cases (92).
The potential role of meat animals in transmission is
less clear and probably less signiŽ cant. Food can become
contaminated by manure from animals that may have active
infections or may be asymptomatic carriers. This was presumably the case in the Nova Scotia outbreak, in which
manure-contaminated coleslaw was implicated (176).
It is likely that common environmental sources serve
as reservoirs of strains that infect both meat animals and
humans by potentially independent routes. The infection of
animals through contaminated feed and poor-quality silage
is well documented (54, 63, 207). The same strains can
cause human illness following their introduction and establishment in food processing facilities through environmental contamination from still unknown sources or via other
vehicles, including food handlers. Systematic data on the
contamination of food handlers are currently lacking. The
fact that certain well-studied virulent clones (e.g., ECI) are
generally rare in processing environments and foods suggests that their entry may be a relatively rare event or that
their establishment in food processing facilities requires
specialized conditions or environmental niches.
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bodies and resistant to serotype 4b–speciŽ c phages. Similar
antibody-negative, phage-resistant strains were identiŽ ed in
both the California and Massachusetts outbreaks (38). Although we cannot exclude the possibility that these phenotypes became established during passage and storage of
the bacteria in the laboratory, an alternative possibility is
that the observed variants were selected during the infection
of their human host. Teichoic acid substituents are strong
immunogens (196), and glycosylation-negative variants
may be at an advantage in terms of immune system evasion.
The characterization of multiple isolates from additional outbreaks may show similar evolutionary, populationlevel events. In fact, such events may account for certain
atypical results reported in the literature. For instance, characterization of the strains implicated in the Swiss outbreak
(1983 to 1987) showed one common MEE type but two
closely related genotypes and phage types. In addition,
identical genotypes were seen in strains that were either
nontypeable by phage typing or of different phage types
(139). Some differences in phage types may correspond to
strains that are deŽ cient in the expression of teichoic acid
substituents, which function as receptors for certain phages
in L. monocytogenes (205). Nocera et al. (139) noted that,
if the phage typing data were to exclude such strains from
the outbreak designation (as indeed was the case), the extent of the outbreak could be underestimated.
1819
3.0. L. MONOCYTOGENES STRAINS IN
FOODS AND IN THE FOOD
PROCESSING ENVIRONMENT
3.1. Origin of food contamination. A clear pattern has
been emerging from numerous studies in different food processing plants, primarily in Europe, during the past 10
years. The primary source of food product contamination
before release to consumers appears to be the processing
environment. The incidence of L. monocytogenes in milk
in processing centers (33.3%) was substantially higher than
1820
KATHARIOU
3.2. Types of strains found in the processing environment. Certain serotypes tend to be prevalent in processing environments and in foods. In an extensive study
of strains contaminating pork slaughtering and cutting
plants, Giovannacci et al. (73) found that the prevalent serotypes were 1/2a, 1/2c, 3a, and 3c. Serogroup 1/2 was
prevalent in several other surveys of processing environments for poultry and processed meats, as well as in Ž nal
products (107, 149, 194).
3.2.1. Resident strains in food processing facilities.
Strain subtyping has shown the presence of both transient
(sporadic) and resident (persistent) strains in the processing
environment. Persistent strains have been found in products
that originated from different producers and were processed
in a common facility (82, 83), suggesting that their source
was the food processing environment itself or a common
ingredient. Such strains can become established in a speciŽ c facility, and the literature contains several reports of
certain strains having been isolated repeatedly over several
years. In fact, the strain implicated in the 2000 multistate
outbreak of listeriosis in the United States involving contaminated deli turkey meats (134) appears to have persisted
in the processing facility for more than 10 years (section
2.5.3).
Even though multiple strain types can be isolated from
a processing plant or from the Ž nal product, one or a few
strains (clones) frequently become established in the facility, with these strains often found in the Ž nished product
as well (107, 115, 148a, 149, 169). The literature contains
numerous additional reports on resident clones in seafood,
dairy, poultry, and pork processing facilities, suggesting
that these strains are common in the food industry. It is
possible that such strains get established in the processing
facilities because they compete effectively against other
bacteria, or against other L. monocytogenes strains, by
means of currently unknown mechanisms (including, but
not limited to, phage resistance, bacteriocin production, biocide resistance, competitive adherence to surfaces, and biofilm formation). Possibly, certain conditions in the process-
ing plant select for the resident clones while inhibiting other
strains.
In foods and food processing facilities, L. monocytogenes often coexists with other Listeriae, especially Listeria
innocua, with the latter frequently outgrowing L. monocytogenes in commonly used selective media (43, 154). Currently, however, we lack an understanding of the possible
interactions between L. monocytogenes and other Listeriae
in situ (e.g., in the processing environment and foods).
3.3. Contribution of persistent strains in the processing environment to foodborne listeriosis. In general,
strain types derived from food processing environments
only partially overlap with those implicated in human illness. This is strongly suggested by the observed serotype
distribution patterns. Many resident clones are serotype 1/
2c, which only rarely causes human illness. Other prevalent
serogroup 1/2 serotypes, however, are not rare among clinical isolates, and additionally, serotype 4b can persist in
food processing facilities, albeit less frequently (see below).
Qualitative and quantitative analysis of strain diversity
and persistence has been most rigorously and extensively
done in seafood and seafood processing plants, as RTE
cold-smoked salmon and other Ž sh products have a relatively high incidence of the pathogen (18). Multiple strains
appear to coexist in Ž sh products (23, 71), and strain clusters associated with speciŽ c areas and operating procedures
in the processing plant have been identiŽ ed. For instance,
one survey of strain types in a shrimp processing plant
showed a discrete strain cluster associated with water and
utensils (53). In another study, strains associated with brining and salting were identiŽ ed in a cold-smoked rainbow
trout plant and in a plant producing vacuum-packed,
smoked, and cold-salted Ž sh products (11, 94).
On several occasions, clones that were persistent and
prevalent in processing facilities were also associated with
human listeriosis (29, 58, 83, 148a). A report of special
interest concerned the only characterized outbreak of invasive listeriosis that involved contaminated Ž sh (trout)
(58). The implicated strain, which was both persistent and
prevalent in the plant, was serotype 4b. In another study,
1/2a, 1/2b, and 4b strains were repeatedly detected as contaminants of soft cheeses, and subtyping of the serotype 4b
strains showed that they were similar to strains implicated
in human illness, including an earlier outbreak (121).
In conclusion, these and other data suggest complex
listerial contamination patterns in the processing plants.
Certain resident clones in the processing plant (e.g., those
of serotype 1/2c) are not commonly implicated in human
listeriosis, whereas others clearly have the potential to cause
human illness, depending on their serotype, strain type, and
level of contamination of the Ž nished product.
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in samples from dairy farms (5.3%) (81). An extensive survey of levels of meat contamination indicated that chilling
and cutting signiŽ cantly increased the contamination of
pork, in agreement with the high environmental prevalence
of the pathogen (71 to 100%) in the chilling-cutting area
of the processing plant (197). Very similar results concerning the impact of chilling rooms were reported in an earlier
study of mutton contamination in New Zealand (160). Although whole-carcass contamination levels of sheep, cattle,
and swine were remarkably low, minced beef was extensively contaminated (21 of 23 samples) (65). Before slaughter, L. monocytogenes was not commonly detected in poultry fecal samples, but poultry became heavily contaminated
after slaughter. In surveys of poultry slaughterhouses, live
birds contributed little to listerial contamination (42a, 148a,
149). Similar results have been obtained from extensive
work in smoked Ž sh plants, where the genotypes in the
plant, or on the Ž nished products, were often different from
those on the raw Ž sh (11, 143, 169).
J. Food Prot., Vol. 65, No. 11
3.4. Comparative virulence of food-derived versus
clinical strains of L. monocytogenes: 3.4.1. Evidence
from epidemiology and subtyping. The epidemiologic and
strain subtyping data have prompted speculation that human
virulence may be attenuated in many, if not most, strains
contaminating food (88, 144).
There is little doubt that clinical serotypes (mostly 1/
J. Food Prot., Vol. 65, No. 11
L. MONOCYTOGENES VIRULENCE AND PATHOGENICITY
3.4.2. Laboratory determinations. The evaluation of
the potential food safety threat posed by many strains contaminating food is complicated by the absence of a laboratory standard for human virulence and the fact that virulence estimates can be obtained only from animal and cell
culture models. Such virulence models have not identiŽ ed
consistent differences between food and clinical isolates of
L. monocytogenes, although interstrain variation within
each of these groups was observed (157, 158). Using two
different cell culture models that allowed quantitation of
invasiveness and cytotoxicity to mammalian cell cultures,
Del Corral et al. (51) determined that no systematic differences could be found between 30 food and clinical strains.
In a larger survey, Conner et al. (39) determined that most
strains were pathogenic in the immunocompromised mouse
model and identiŽ ed only a small number of weakly hemolytic strains with attenuated virulence. A rigorous
screening of a large collection by Brosch et al. (30) revealed that only 2 of 63 food strains were avirulent in mice,
leading the authors to conclude that all strains posed potential health hazards, regardless of their source, serotype,
or genotype. Screening a smaller number of strains with the
mouse bioassay as well as the chick embryo test, Notermans et al. (147) concluded that ‘‘almost all serovars present in food have clear virulence properties.’’ In another
study employing the chick embryo model, strains of different MEE types of both food and clinical origin were
pathogenic, but death of the chick embryos occurred more
rapidly following infection by clinical versus food-derived
strains (141).
Although these investigations all suggest that L. monocytogenes isolates from food are basically virulent in the
models that were used, several other studies indicate that
possible differences between food and clinical strains can
be identiŽ ed using other experimental designs. It is not
clear whether such differences are ‘‘indicators’’ of virulence
or representative of actual virulence characteristics. Facinelli et al. (61) found that a signiŽ cant fraction (16 of 23)
of food-derived strains (but none of the 12 screened clinical
strains) were negative with a panel of lectins, which normally bind to sugars on the cell surface of the pathogen.
Interestingly, the lectin-negative strains were avirulent in a
cell culture model for virulence. In a comparison of clinical
and meat-derived strains, Avery and Buncic (13) found that
freshly grown isolates were indistinguishable in their virulence in the chick embryo model; however, signiŽ cant differences could be observed after the inoculum had been
maintained in buffer at 48C. Clinical strains remained virulent, whereas the virulence of several meat-derived strains
was noticeably reduced. In addition, meat-derived strains
had a longer lag phase when shifted from 4 to 378C (34).
Even though a relatively small number of strains from
one source (meat) were included, these latter results are of
interest, considering that foodborne listeriosis commonly
involves refrigerated foods. Although the physiology of the
transition of L. monocytogenes from the cold, starved state
to growth at 378C in rich media is far from clear, interstrain
differences likely exist. Furthermore, such studies suggest
the possible impact of environmental modulation on virulence. It is possible that the cold environment associated
with foods and food processing facilities, along with other
existing stresses, selects for special subpopulations of
strains with adaptive physiology attributes that promote
both survival in the food and human virulence. These subpopulations, representing only a minority of strains in food,
would serve as inocula for humans and would eventually
become represented among clinical strains.
In conclusion, the available data indicate that some
food-derived strains of L. monocytogenes may have surface
antigenic, genetic, and physiologic characteristics that can
differentiate them from the majority of human clinical
strains but do not provide conclusive evidence for differences in human virulence between clinical and food-derived
strains.
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2a, 1/2b, and 4b) do not accurately re ect the serotypes
prevalent in foods and food processing facilities (1/2a, 1/
2c, 3a, and 3c). However, substantial evidence indicates that
at least a fraction of the different strains in dairy, meat, and
poultry products genotypically overlap with strains causing
human illness (24, 64, 92, 121, 172, 193). In the study by
Ryser et al. (172), the relative recovery of ‘‘clinical’’ (i.e.,
subtypes related to those associated with human illness) and
‘‘nonclinical’’ subtypes from meat and poultry products
was dependent on the enrichment protocol used, suggesting
that these distinct strain types are physiologically different
as well. The extent to which food-derived strains, as a
group, may be genetically distinct from clinical strains
needs to be rigorously deŽ ned. A recent polymerase chain
reaction–based typing investigation using the distribution of
repeated sequences in the genome suggested a distinct genomic group for food-derived versus clinical strains of L.
monocytogenes (93), but the surveyed collection of food
strains consisted of a rather small number of independent
isolates.
1821
3.5. IdentiŽ cation of virulent food strains of L. monocytogenes: 3.5.1. The role of new and improved models.
If, indeed, certain food strains are more likely to cause human infection than others, the question arises as to whether
and how such problem-prone strains can be identiŽ ed. Experimental identiŽ cation with high discriminatory potential
would be extremely valuable but is fraught with problems
intrinsic to method design and the speciŽ c experimental
model that is used. Animal and cell culture models may
never be completely satisfactory because not all components of human foodborne infection can be realistically simulated in these models. The search for new and better animal models continues. For example, work is currently under way on new animal models (including transgenics) that
are more relevant to oral infections (e.g., (109)). It remains
to be seen if this is a cost-effective investment in terms of
relevance of the results to human foodborne disease.
Valuable information can be obtained from carefully
designed, compartmentalized model systems chosen specifically for their relevance to human infection (e.g., the infection and activation of human vascular and brain endothelial cells, which provide indications as to the potential
1822
KATHARIOU
of the bacterial strain to induce an in ammatory response
and to pass through the blood-brain barrier). Such systems
have been established primarily for immunological and genetic studies (57, 80, 102, 210). These systems have not
yet been used to evaluate different strains from food, but
they hold promise in this regard.
which is now commonly required before a new gene can
be described in a publication. Participation must be clearly
voluntary for the food industry, with appropriate protections instituted for conŽ dentiality.
3.6. Virulence-attenuated strains of L. monocytogenes in foods: possible protective roles? In animal models,
strains with attenuated virulence persisted for short periods
in the animal and induced protective immunity against
pathogenic strains when administered in sufŽ cient doses
(37). The consumption of foods contaminated by strains of
relatively low virulence may cause subclinical infections
that similarly protect humans against more virulent strains.
Presuming that the putative foodborne-attenuated strains
share key antigenic determinants with the virulent strains,
the potential for antibodies or other immune responses to
the former to confer protective immunity toward the latter
may be substantial. Such cross-protection would especially
apply for strains in the same serogroup, which share immunodominant sugar substituents on their cell surface. Interestingly, in each major serogroup, some serotypes are
more commonly encountered in illness than others. In the
case of serogroup 4, strains other than 4b had reduced virulence in animal and cell culture models (184). It is possible that subclinical infection by such strains may confer
resistance to infection by serotype 4b.
Perhaps of greater interest is the observed frequent
contamination of foods by strains of serotype 1/2c, which
is rarely implicated in illness. All 1/2c strains screened to
date are genetically closely related (36, 138) and harbor a
mutation in one of the virulence genes (95), suggesting that
this group of strains may have arisen rather recently. Conceivably, the contamination of foods by serotype 1/2c may
render humans more resistant to infections by virulent
strains with similar surface antigens, e.g., serotypes 1/2a
and 1/2b.
The potential for L. monocytogenes to cause mild disease, which in many cases goes undiagnosed, has been reported (168). However, the extent to which humans, including immunosuppressed individuals, may be infected by
L. monocytogenes asymptomatically, or with only mild
symptoms, has not been rigorously determined. It will be
of interest to determine the possible correlations between
the exposure to foods contaminated by clinically rare strains
(e.g., serotype 1/2c), the carriage of L. monocytogenes, and
the incidence of foodborne listeriosis in distinct cohorts
chosen in terms of dietary habits, immunodeŽ ciencies, and
other risk factors. The identiŽ cation of humans who have
had an immune response to L. monocytogenes is currently
difŽ cult, as antibodies are frequently of low titer and commonly cross-react with antigens from other gram-positive
bacteria.
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3.5.2. Biotype- and genotype-based predictive assessment of virulence. General predictive assessments of
human virulence can be made on the basis of certain wellestablished bacteriologic criteria, such as phage type and
serotype. Strains of certain serotypes (e.g., 1/2c, 4a, 4c, 4d,
and 4e) are rarely recovered from patients and may constitute a relatively low (but not negligible) risk for human
listeriosis. Following the same reasoning, the presence of
serotype 4b in an RTE food should be cause for concern
because, while not commonly prevalent in foods, such
strains have been involved in numerous outbreaks. The risk
associated with such contamination would be substantially
increased if molecular subtyping data suggested that the
strain belonged to a genotype common to one of the known
epidemic-associated clones.
Strains of serotype 1/2a contribute substantially to human listeriosis and are, in addition, frequently found in
RTE foods and food processing environments. Assessing
the risk posed by food contamination by bacteria of serotype 1/2a is especially challenging: serotype 1/2a has a relatively high genetic variation, and the identiŽ cation of 1/2a
lineages that are predominant in human illness has not yet
been achieved. The need exists to augment the typing database for strains of serotype 1/2a, both from foods and
from clinical cases.
Such a system is in place through PulseNet for clinical
isolates and for food isolates provided by government laboratories, but it must be rigorously extended to include additional food isolates. Currently, food microbiologists in the
academic and industrial sectors who isolate and type L.
monocytogenes do not have access to PulseNet. Hence, a
large portion of the food strain genotype data is being underutilized. Furthermore, the food strain genotype database
should not be limited to strains derived from North America. Food contamination is an international issue, and the
incidence of outbreaks due to ECI makes it abundantly
clear that this bacterial clone knows no national borders. In
addition, a review of the literature has revealed that most
large-scale, rigorous studies on L. monocytogenes genotypes isolated from foods and food processing plants are
being conducted outside North America, especially in the
Scandinavian countries and elsewhere in Europe.
A continuously augmented standardized international
database based on the PulseNet format but accessible to
scientists from government, industry, and academic institutions will greatly facilitate the identiŽ cation of food lineages that are also encountered in infection and will, in
addition, determine the proportion of the food strains that
may constitute relatively low risks for human listeriosis.
Participation in this database should be expected for academic and government investigators, analogous to the entry
of nucleotide sequence data in an accredited database,
J. Food Prot., Vol. 65, No. 11
4.0. A VIEW FOR FUTURE NEEDS
4.1. Theoretical studies and modeling. Almost all reported studies cited in this review clearly point to the food
processing environment as the most relevant target for interventions that will reduce the incidence of Listeria in
foods. A clear need currently exists for theoretical tools that
J. Food Prot., Vol. 65, No. 11
L. MONOCYTOGENES VIRULENCE AND PATHOGENICITY
4.2. Additional microbiologic and epidemiologic
studies. Additional surveys are needed to understand the
contribution of other segments of the farm-to-fork continuum to the burden of foodborne listeriosis. Examples include food service settings such as restaurants, salad bars,
and delicatessens as well as consumer preparation and handling in the home. Household pets can reportedly shed and
carry L. monocytogenes without showing symptoms, as can
household members who are asymptomatic carriers of the
organism. We should assess the contribution, if any, of
these sources to the burden of human illness. In addition,
special attention should be given to the ecology of lineages
that are highly relevant epidemiologically, such as epidemic
clones and serotype 4b strains, for the identiŽ cation of currently unknown reservoirs and ampliŽ cation niches.
4.3. Construction of a strain library for an augmented typing database. We need an augmented PulseNet
(or equivalent) database for foodborne pathogens that
would have the infrastructure and resources needed to make
it truly accessible to all accredited, properly trained, and
properly equipped microbiologists in government, industry,
and academic sectors. This would greatly facilitate the
monitoring of food and environmental contamination
throughout the country and would also allow the incorporation of comparative data from scientists in other countries. The importance of such an endeavor for enhancing
food safety and public health cannot be overemphasized.
We also need the active participation of industry in
establishing a collection of strains speciŽ c to processing
environments, so that strains and species from processing
microhabitats can be adequately represented. Such a plan
should include protocols to safeguard participating industry
members from any putative actions arising from strains or
species submitted to the collection.
4.4. Genomics and proteomics: expected impact.
The currently available typing tools, including serotyping
and molecular subtyping, largely fail to provide adequate
insight into strain virulence and the likelihood that any given strain will cause illness. The reasons are primarily that
the resolution of these tools is relatively limited and relevant only to selected small regions of the genome, which
are examined without reference to the rest of the organism’s
genetic endowment and adaptive potential. With the completion of the complete genome sequence determination of
L. monocytogenes (strain EGD) (74), a new generation of
genome-level tools is becoming available for strain typing,
including, but not limited to, DNA chips (microarrays).
Gene microarrays will permit whole-genome comparisons
among diverse strains and the identiŽ cation of strain- or
lineage-speciŽ c sequences. The hybridization of DNA microarrays with genomic DNA from other strains (genomotyping) will identify genomic regions of divergence in different strains and will provide rigorous estimates of genetic
variation in distinct lineages, as is being already done with
other pathogens (55, 173). Furthermore, expression microarrays will be utilized to determine the expression pattern of the organism’s genes (representing all or a selected
fraction of the genome) in response to speciŽ c conditions.
For typing purposes, differences in such multigene or genome-level expression proŽ les among different strains may
be especially useful. Expression proŽ les speciŽ c for strains
that are of special epidemiologic relevance (e.g., strains implicated in epidemics) may be determined. With the support
of adequate epidemiologic data, virulence-related genomic
signatures (at the DNA sequence or gene expression level)
are expected to emerge from analysis of the genomes. Such
tools, along with other DNA sequence–based typing tools
(e.g., multiple-locus sequence typing), will allow the rapid
screening of isolates relative to the constellation of genetic
markers characteristic of virulent strains of diverse serotypes as well as those characteristic of especially troublesome lineages, such as those implicated in outbreaks.
Contributions are also expected from proteomic analysis of the organism’s complete protein signatures. Current
methodologies such as matrix-assisted laser desorption-ionization mass spectrometry hold promise not only for typing
purposes but for the identiŽ cation of expression patterns
unique to selected strains or lineages. Strain- or lineagespeciŽ c proteins are likely to be identiŽ ed, and additional
insight will be obtained from the identiŽ cation of proteins
that have unique expression patterns (e.g., timing of ex-
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will analyze the available data on Listeria prevalence in
food processing facilities and in foods with enhanced analytic and predictive potential. There is especially a need for
modeling the niche complexity of Listeria in the food processing facilities. To be effective, such modeling would
have to be speciŽ c to the processing facility in terms of the
product that is being processed (type, volume, and other
key variables). ‘‘Complexity measures,’’ as both a concept
and a formalism, are already common in certain branches
of the new mathematics (17, 201), and such measures as
these could be adapted to a more formal description of the
many bacterial niches within the food processing environment. Such studies will enhance our ability to understand
and subsequently control the incidence of Listeria in food
processing environments.
Such models would serve as good ‘‘costing tools’’:
they would help deŽ ne the facilities’ speciŽ c maintenance
needs and critical control points, thereby facilitating the estimation of the Ž nancial resources that would be required
for any corrective action.
Important contributions could be made by theoretical
studies that address outcomes on the basis of interactive
processes (e.g., between microorganisms in a bioŽ lm or between microbial cells and a surface), as well as by any
studies that address linked and repetitive processes. The
conclusions from such studies should be formulated so that
they are meaningful to both the industrial user and the microbiologist. Effective model construction can only be delivered by teams that include applied mathematicians as
well as microbiologists. A regular, periodic (e.g., every 5
years) literature review of models such as that proposed
should be undertaken, again by individuals with combined
expertise in applied mathematics and microbiology, and
should focus on the industrial user and the processing plant
microbiologist.
1823
1824
KATHARIOU
J. Food Prot., Vol. 65, No. 11
pression, stability, and amount). It will be essential that
strains of special epidemiologic and food safety relevance
are adequately represented in such genomic and proteomic
studies.
15.
ACKNOWLEDGMENTS
16.
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