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Listeria monocytogenes: General Overview and Its Signiﬁcance to Food

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roman_LESTERIA.indd 1 6/30/16 1:18 PM
Mohd Nizam Lani
Zaiton Hassan
Penerbit UMT
Universiti Malaysia Terengganu
21030 Kuala Terengganu
Terengganu
2016

Listeria monocytogenes: General Overview and Its Significance to Food Safety
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Includes index
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TABLE OF CONTENT
Preface vii
1 WHY Listeria monocytogenes IS AN IMPORTANT

FOODBORNE PATHOGEN? 1
2 GENERAL CHARACTERISTICS OF L. monocytogenes 3
3 FACTORS AFFECTING GROWTH AND SURVIVAL OF
Listeria sp. IN FOODS 15
4 THERMAL INACTIVATION OF L. monocytogenes 19
5 STRESS AND EFFECTS ON VIRULENCE OF L. monocytogenes 33
6 CURRENT METHODS FOR CONTROLLING L. monocytogenes
IN THE FOOD INDUSTRY 37
Bibliography 41
Index 57

vii
PREFACE
Foodborne pathogens continue to cause major public health problems worldwide.

One of the most significance of foodborne pathogenic bacteria within the last three
decades is Listeria monocytogenes, which continue to cause sporadic cases and
outbreaks of illness linked to consumption of food products. This publication briefly
guides the readers on the general characteristics of L. monocytogenes in the nature
of taxonomy, isolation and detection in foods, reservoirs and occurrence in food,
epidemiology and diseases. As L. monocytogenes is ubiquitous in nature, human,
animals and environment serve as primary reservoirs of this organism. Therefore, it is
not surprising that any fresh food product from animal and plant origin may contain
L. monocytogenes through poor fertilisation methods and lack of hygiene practices
in agriculture.
Listeria can grow and survive in many different foods. The growth and survival of
L. monocytogenes in foods is governed by a variety of environmental and ecological
factors. The success of L. monocytogenes as a foodborne pathogen is strongly
influenced by its ability to survive under wide adverse environmental conditions.
These include temperature, pH, acid, salt, water activity and modified atmosphere.
Understanding the factors affecting the growth and survival of L. monocytogenes
in foods may provide principles of preservation strategy that can be used by food
industry.
This publication highlights on thermal inactivation of L. monocytogenes.

Temperature plays an important role to control and eliminate this organism in the
food industry. This publication describes the theory death of microbes by heating that
determine the heat resistance of bacteria at a constant temperature using D-value.
Heat resistance of L. monocytogenes in foods are described deliberately. In addition,
this publication also addresses on the sublethal injury at high and low temperature
stress of this organism. The recovery of sublethal injury of L. monocytogenes is also
discussed.
As intracellular pathogen, L. monocytogenes is able to multiply in the most

environments exploited by extracellular parasites. This publication briefly describes the
effects on virulence determinants in L. monocytogenes upon exposure to a particular
stress response. The complex interaction between survival and multiplication of this
bacterium within host tissue will affect the disease manifestation.

viii
On the last chapter, this publication addresses on current methods for controlling
L. monocytogenes in the food industry. In food industries, various means of controlling
and minimizing contamination of L. monocytogenes through hurdle technology,
natural antimicrobials from microbes, plants and animals, food irradiation, high
hydrostatic pressure treatment, pulsed electric field treatment, ultrasound treatment
and ultraviolet light treatments.
Overall, this publication serves as an introductory resource for those interested

in L. monocytogenes. In fact, this publication can be used as an essential reference
for food microbiology course for undergraduate level. This publication also can be
used as an essential guide for anyone in the food industry, research or regulation
related to food safety issue and enforcement. This publication is aiming to enhance
the awareness of food safety issue as a part of continuing global effort to ensure a
safer and better world for now and future.

CHAPTER 1
WHY Listeria monocytogenes IS AN IMPORTANT

FOODBORNE PATHOGEN?
1.1 INTRODUCTION
Food-borne infections still remain as one of the important causes of preventable

morbidity and mortality in the world although a lot of advanced knowledge,
technologies and innovations have been developed in the 21st century. In the
effort of reducing foodborne illness cases, the government and food industry do not
have simple solution on this matter. Indeed, foodborne illness involves a complex
interaction of factors that need to be addressed and the solution must come from
collective and collaborative efforts from all parties involved. The ever-changing nature
and sophistication of microbial food safety require strategic and dynamic control
methods to sustain the level of safety and increase the quality of food products (IFT
Expert Report, 2002).
There are many traditional methods for preservation have been used in the food
industry, such as controlling the water activity, pH and heat treatments, chilling
and freezing of foods. The keys of food preservation are principally based on one
of three ways; (i) preventing access of microorganisms to foods; (ii) inactivating
microorganisms if they have accessed into foods; and (iii) preventing or slowing
down the growth of microorganisms (Gould, 2000). The heat treatment used to kill
microorganisms and inactivate enzymes as part of preservation strategy may have
detrimental effects on the nutritional and sensory attributes of the products (San
Martin et al., 2003). Therefore, finding the alternative methods for preservation is
critical as the demand for minimally processed foods that are safe, nutritious and
fresh have been increasing (Sloan, 1999).
The success of Listeria monocytogenes as a foodborne pathogen is highly

influenced by its ability to survive under a wide range of environmental conditions.
Thus, its presence in food may cause a serious health hazards to consumers. This
psychrotrophic bacterium is ubiquitous in nature and has been linked to several
number of outbreaks of foodborne illnesses. This bacterium can cause serious
diseases, particularly during pregnancy and people with weakened immune system
(Farber & Peterkin, 1991;2000). From 1998-2015, based on report by Foodborne
LISTERIA.indd 1 6/30/16 1:09 PM

2 / Listeria monocytogenes: General Overview and Its Significance to Food Safety
Outbrek Online Database (FOOD Tool), the outbreaks caused by Listeria were 18,
211 outbreaks in the United States. (CDC, 2015).
When environmental parameters are significantly different from the microbial

optimal range, the microbes may undergo stress. These stresses that occur naturally
in many foods, or are deliberately imposed on foods have influenced on the food
preservation and safety assurance of foods. This is an aspect of food preservation
strategies that can have significant effects on microbial physiology, including changes
in stress resistance, cell morphology and expression of virulence genes (Rowan,
1999).
Exposure to a particular environmental stress may invoke a stress response

which not only enables the bacterium to better resist to that particular stress, but may
also provide enhanced cross-protection against other stresses (Lou & Yousef, 1997).
This ‘stress hardening’ phenomenon that refers to the increased resistance to lethal
factors after adaptation to environmental stresses may affect the effectiveness of food
preservation hurdles and compromise food safety. Besides the increased resistance
to inactivation associated with food processing, stress-hardened pathogens may also
have increased virulence, since many pathogens are able to sense environmental
stresses as signals for expression of virulence genes.
Traditional methods of pasteurisation or sterilisation using thermal or chemical

treatments have generally been effective but nevertheless they have certain
limitations, such as the emergence of pasteurisation-resistant bacteria. There is
currently considerable interest in alternative methods for the control of problematic
microorganisms. The non-thermal treatments such as microwave and radio wave
processing, high pressure processing, ozone and plasma treatments, ultra-violet
(UV) light treatment, pulsed ultra-violet (UV) treatment, pulsed electron beam and
X-ray treatment, pulsed corona treatment, arc discharge treatment and pulsed
electric field treatment have potent anti-microbial properties that effective in
reducing undesired microorganisms (Espie et al., 2001).
This publication provides brief background on L. monocytogenes as an important
foodborne pathogen, and describes the factors influencing the growth of this organism
in foods. This is important because temperature, pH, acid, salt, water activity and
gaseous environment has significant influence on the survival of this organism in
food. This publication continues to focus on thermal inactivation of this organism as
temperature plays an important factor to control the growth of this organism. Then,
stress and the effects on virulence of L. monocytogenes is briefly discussed. Finally,
the publication emphasizes on the current technologies to combat this pathogen in
the food industry.

CHAPTER 2
GENERAL CHARACTERISTICS OF
Listeria monocytogenes
2.1 Taxonomy of L. monocytogenes
Listeria monocytogenes is an individual from the genus Listeria and is characterized as

a Gram-positive rod,, non-spore former, facultatively anaerobic which is a part of the
lactobacilli family alongside genera, for example, Lactobacillus and Streptococcus
(Rocourt, 1999). They are regular rods 0.4 X 0.5 µm by 0.5-2.0 µm with rounded
ends, are sometimes almost coccoid, occurring singly or in short chains and less
often in long filaments (Sutherland & Porritt, 1997). They are motile by a couple of
peritrichous flagella, best communicated at 20-25°C. A culture stab in semisolid
growth medium produces growth along the stab line, spreading horizontally 3-5
mm below the surface in an umbrella pattern (Ryser & Marth, 1991). G+C content
of the DNA of Listeria from 37 to 39 mol% and is firmly identified with the Bacillus,
Lactobacillus, and Streptococcus genera. From 16S ribosomal RNA (rRNA) data,
Listeria was nearest to Brocothrix, Staphylococcus and Kurthia (Jones, 1988).
The genus Listeria is classified in Bergey’s Manual of Determinative Bacteriology

under the “Regular, Non-sporing Gram Positive Rods”, in a section under Group 19
(Holt et al., 1994). Genera of Group 19 are rod- shaped (varying from short, almost
coccal, rods to elongated rods, filaments, or trichomes) but exhibit a regular shape
with little pleomorphism. They are Gram positive, non-sporing, seldom pigmented,
mesophilic, and chemo-organotrophic, and they grow only in complex media. They
are generally connected with plants or creatures or with rotting natural matter; a
few genera contain pathogens (Holt et al., 1994). The differentiation of Listeria from
similar genera is compressed in Table 1.

Table 1: Differentiation of Listeria from similar genera

(Varnam and Evans, 1991; Seeliger and Jones, 1986)
Property Listeria Brochothrix Lactobacillus Kurthia

Blue-green colonies1 + -2 - -
Facultative anaerobe + + + -
Motile at 25°C + - +3 +
Growth at 35°C + - 4
+ 5
+
Acid from glucose + + + -
Catalase + + - 6
+
1
When viewed microscopically using oblique lighting on tryptose agar
2
There are unconfirmed reports that some strains of Brochothrix have a similar colonial
appearance to Listeria
3
Strains of some species may be motile on high pH value, low carbohydrate media
4
Occasional strains of Brochothrix-like bacteria will grow at 35°C
5
Some strains, especially from meat, will not grow at 35°C
6
Strains of some species may give a positive reaction on a high pH value, low
carbohydrate medium, or in the presence of haem
The genus Listeria was previously classified among “genera of uncertain

affiliation” (Seeliger & Jones, 1986) was initially portrayed by Pirie as monotypic
and contained only L. monocytogenes. In 1985, although eight Listeria spp. namely
L. monocytogenes, L. ivanovii, L. innocua, L. welshimeri, L. seeligeri, L. denitrificans,
L. murrayi, and L. grayi were perceived in the Approved Lists of Bacterial Names in
the ninth edition of Bergey’s Manual of Systematic Bacteriology, where three Listeria
spp. namely L. grayi, L. murrayi, and L. denitrificans have been relegated to a different
taxonomic status as species incertae sedis (species of uncertain position) (Jones
& Seeliger, 1986). However, a low level of 16S ribosomal RNA content between
L. denitrificans and other Listeria spp. (Stuart & Welshimer, 1973) proposed to be
exchanged to another variety Jonesia as Jonesia denitrificans (Rocourt et al., 1987).
2.2 Identification of Listeria Species

Distinctive plans for confirmation have been built up which utilize various
morphological and biochemical tests. Early screening for Listeria spp confirmation
and speciation of L. monocytogenes involves three following microbiological tests
such as motility, hemolysis and three carbohydrate agars (mannitol, rhamnose and
xylose). All Listeria spp. except L. grayi and L. murrayi should be mannitol-negative

General Characteristics of Listeria monocytogenes / 5
as only these two species can utilise mannitol. On the other hand, L. murrayi is the
only species, which can reduce NO3- to NO2-. L. monocytogenes, L. ivanovii and
L. seeligeri produce hemolysis in horse or sheep blood agar. It shows positive in
the CAMP test. Of the three, only L. monocytogenes cannot utilise xylose, but is
rhamnose-positive. L. ivanovii can be differentiated from L. seeligeri by the CAMP
test, where L. seeligeri shows enhanced hemolysis only at the Staphylococcus streak
and L. ivanovii shows enhanced hemolysis in the area of the R. equi streak. L. innocua
can only be differentiated from L. monocytogenes by its lack of hemolysis on blood
agar plates and negative reaction in the CAMP test. L welshimeri that is rhamnose-
negative may be confused with a weakly-hemolytic L. seeligeri unless the CAMP test
is run (Pagotto et al., 2001). L. monocytogenes might be separated from different
types of Listeria as indicated by the criteria in Table 2.
To confirm the purity of the isolates, the biochemical and morphological tests
were performed. An isolate was inoculated into Tryptone Soya Broth–Soybean Casein
Digest Medium USP with 0.6% Yeast Extract (OXOID, UK) prior to subculturing
into Listeria Selective Agar with Listeria Selective Supplement (Oxford Formulation)
(OXOID, UK) and Blood Agar Base added with 7% sterile sheep blood (OXOID,
UK). It was then incubated at 37°C for 24 to 48 hours. Presumptive positive Listeria
colonies were grey green with a black sunken centre, and exhibited a black halo in
Listeria Selective Agar with Listeria Selective Supplement (Oxford Formulation) after
incubation period hour of 24-48 at 37°C. The colonies were picked up and further
purified by streaking onto Tryptone Soya Agar with 0.6% Yeast Extract (OXOID, UK).
The utilization of blood agar permitted observation of ß-haemolysis. ß-haemolysis is
caused by a complete lysis of the red cells in the medium.
Isolates that are Gram (+) as shown in Figure 1, catalase (+), oxidase (-), motile
at 21°C, methyl red (+), ß-haemolysis (+), reduction of nitrates (-), rhamnose (+), and
xylose (-) are considered to be L. monocytogenes (The Oxoid Manual, 1998). Further
confirmation using a Listeria API Kit is carried out and the results of biochemical tests
are as follows: DIM (-), ESC (+), αMAN (+), DARL (+), XYL (+), RHA (+), MDG (+), RIB(-
), G1P(-), TAG(-), and ß-haemolysis (+). A streak plate culture of L. monocytogenes on
blood agar is displayed in Figure 2 while the conventional differentiation of Listeria
spp. is summarised in Table 2 and comparison between conventional and alternative
techniques for the confirmation of Listeria spp. and L. monocytogenes is displayed
in Table 3.

Figure 1: Gram staining of L. monocytogenes cells
Figure 2: Single colony of L. monocytogenes on Blood Agar

Table 2: Conventional differentiation of Listeria spp.
(Linnan et al., 1988; Jemmi and Stephan, 2006)

Species B-hemolysis Production of acid from CAMP reaction on sheep
blood with
D-Mannitol L-Rhamnose D-Xylose S. aureus R. equi
L. monocytogenes + - + - + -
L. innocua - - V - - -
L. ivanovii ++ - - + - +
L. welshimeri - - V + - -
L. seeligeri (+) - - + (+) -
L. grayi - + - - - -
++: strong positive reaction; +: positive reaction; (+): weak positive reaction
-: negative reaction; v: variable reaction
Table 3: Some practical aspects associated with the use of conventional and alternative
methods for the detection and identification of Listeria spp. and L. monocytogenes (Bell and
Kyriakides, 2005)
Test Presumptive / Futher test Time to carry out Time total time to get Time total time to get
Confirmed result the specific test* positive result from negative result from initial
initial test sample test sample
preparation* preparation*
Conventional: Presumptive Yes 4 days 4 days 6 days
broth
enrichment and
plating
Biochemical Confirmation / No 24 hours 6 days 6 days

tests+ identification of a
purified isolate
Immunoassay/ Presumptive Yes 45 minutes to 2 32-50 hours 5-6 days

ELISA hours
Latex immuno- Presumptive Yes <5 minutes 48 hours 6 days
agglutination
Electrical Presumptive Yes Up to 30 hours 54 hours 6 days
methods
Nucleic acid Confirmed No 45 minutes to 3 48-96 hours 48-96 hours
hybridization hours
Polymerase Confirmed No 5 hours 24 hours 24 hours
chain reaction
* Depends on the specific test used and includes time which may be required for any associated culture
work, e.g. prior to the test, purification of suspect positive cultures and the confirmatory tests.
+ Some biochemical test systems require additional tests to be carried out prior to, or alongside, the
biochemical tests, and all results obtained are used in the identification of the isolate.

2.3 Isolation and Detection of L. monocytogenes

Regulatory and food industry agencies undergone a major problem for the detection
and enumeration of L. monocytogenes in foods. Despite of numerous plating methods
been developed for this purpose, not even one medium has risen to be ideal for all
foods. Efficacy of a medium to allow recovery and enumeration of L. monocytogenes
relies on upon the physiological state of the cells, the type of food being analyzed,
and the presence of containing other competing microflora (Brackett et al., 1990).
In general, foods which contain high population of microorganisms other than L.
monocytogenes require most particular media for direct plating methodology. Less
inhibitory media are essential while investigating foods, which contain harmful cells
of L. monocytogenes (Brackett et al., 1990).
U.S. Food and Drug Administration (FDA) and U.S. Department of Agriculture
Food Safety and Inspection Service (USDA-FSIS) methods are two basic strategies
that have been broadly acknowledged for routine isolation of Listeria in food
and maintenance of handling situations. The FDA technique for the detection of
L. monocytogenes use an enrichment methodology. A 1:10 dilution is made in
enrichment broth, homogenized and incubated at 30°C for 48 hour. Samples are
removed at 24 and 48 hours. It is then being streaked for isolation on Oxoid medium
(OM) and LiCl-phenylethanol-moxalactam agar (LPM) and incubated for 24-48
hours at 35°C on OM or 30°C on LPM. L. monocytogenes colonies on OM are
black with a black halo whilst using indirect illumination, colonies on LPM appear
blue or white. Purified suspect isolates are identified using biochemical tests and
CAMP tests. The USDA-FSIS method differs from the FDA procedure primarily in the
selective enrichment and plating medium used, along with the size of the sample
(Martin and Fisher, 2000).
The circumstance with specific plating media is like that, with enrichment broths
in, that a wide variety of media using a wide range of antimicrobial compounds.
These media vary in their selectivity, and there is proof that there is variety in relative
performance as indicated by the way of the food test.
Strategies proposed by some standard associations and other national bodies

depend on pre-enrichment. Nonetheless, pre-enrichment is completely precluded
in ISO 10560 in 1993 or where inhibitors are added after a few hours of incubation
(Certis, 2000). A negative result might be confirmed in four days, while with different
techniques, a length of five days are required before the result can be declared
negative. Presumptive positive results, as showed by typical colonies on selective

agars, may be obtained after two days with FDA method, but further confirmatory
tests are still required (Curtis, 2000). Recently, latest isolation procedures of L.
monocytogenes detection has been developed by Thermo Scientific (2012) as described
in Figure 3.
Figure 3: Testing Protocol for L. monocytogenes in Most Food Types

(Bacteriological Analytical Manual (BAM), US Food and Drug Administration, 2015).

2.4 Reservoirs of L. monocytogenes

Human, animals and environment serve as primary reservoirs of L. monocytogenes.
Human may contained this organism without symptoms as a result of colonisation
of this bacterium in the intestinal tract. Gaillard et al. (1987) suggested that the
intestinal tract may well be the primary site of entry for L. monocytogenes. The
widespread occurrence of Listeria antibodies in humans without a history of disease
may be explained by carrier state, whether transit or permanent. Patients with
listeriosis often excrete high populations of Listeria cells specimens from 21% of
patients had ≥104 CFU/g of faeces, and 18% of household contacts of patients with
listeriosis fecally shed the same serovar and isoenzyme profile of L. monocytogenes
as the corresponding index case (Jensen, 1993). The role of healthy carriers in the
epidemiology of listeriosis is unclear and warrants further study.
L. monocytogenes is ubiquitous. It has been found in association with a wide

variety of fish, birds, and animals, including sheep, cattle, goats, pigs, horses, geese,
gulls, ducks, pigeons, turkeys, chickens, partridge, eagle, parrot, canary, owl, rat,
moose, fox, lemming, rabbit, ferret, hare, guinea pig, chinchilla, skunk, mink, dog,
cat, deer, raccoon, mouse arthropods, fish, insect larvae, fogs and snails (Gray &
Killenger, 1966). The organism is also widely distributed in the environment and has
been isolated from a variety of sources including soil, vegetation, fecal materials,
silage, sewage, water, green plant material, decaying vegetation and animal feed
(Gray,1963).
2.5 Occurrence of L. monocytogenes in Food

Any fresh food product of animal or plant origin may harbour varying numbers
of L. monocytogenes. In general, the organism has been found in raw milk, soft
cheeses, fresh and frozen meat, poultry, seafood products, fruits and vegetable
products (Sutherland and Porritt, 1997). The greatest threat of contamination with L.
monocytogenes come from ready-to-eat products which do not require further heating
before consumption (FDA et al., 2001). Based on the surveys undertaken by the
Public Health Laboratory Service in the UK showed varying levels of contamination
by L. monocytogenes of certain “high-risk” foods such as soft cheeses (14%) and raw
chicken (60%), ready-to-eat poultry (12%), chilled meals from retail outlets requiring
reheating before eating (18%) and main course items from cook-chill units (2%) (Pini
& Gilbert 1988; Gilbert et al., 1989).
In Malaysia, Arumugaswamy et al. (1994) examined 234 food samples, consisting

158 and 76 samples of raw and ready to eat foods, respectively. The study shows

significant observation in which 22 of the 76 samples of RTE (ready-to-eat) foods

were positive for L. monocytogenes. The detection of L. monocytogenes in a high
proportion of RTE foods indicates that the community at large has been extensively
exposed to Listeria’s threat. L. monocytogenes was also isolated from 3/25 samples of
salted fish (Endang et al., 1998). The extent of Listeria contamination in food suggests
that human listeriosis may be prevalence in the Malaysian community. It is important
for food processors and regulatory agencies to design effective strategies to prevent
growth and survival of L. monocytogenes commonly found in the environment,
including food processing, distribution, and retail environments, in foods, and in
home (Gulam, 2002).
Foods implicated in outbreaks of listeriosis have included products from all of

the major groups: dairy, meat, vegetables and fish/shellfish. Analysis of foodborne
outbreaks indicates that foods that are far more likely to be implicated in a listeriosis
outbreak than others are as follows: (i) use of raw ingredients not subjected to a
listericidal process or products susceptible to post- process contamination, (ii)
refrigerated storage, (iii) product formulation allowing growth of L. monocytogenes,
(iv) extended shelf life (> 10 days), (v) food is ready-to-eat, and (vi) consumption by
vulnerable groups (Bell & Kyriakides, 2005).
2.6 Epidemiology of L. monocytogenes

Investigations of outbreaks have provided the knowledge of the etiology of this
disease organism, particularly in relation to isolation of L. monocytogenes from both
the patient and the implicated food. The most prominent outbreak was in 1985
(Linnan et al., 1988) when L. monocytogenes caused miscarriage in pregnant women
after consumption of Mexican-style cheese in California. 81 women were affected
with 48 deaths (including 19 fetal and 10 neonatal).
Most cases of human listeriosis occur sporadically although much of what was
known about the epidemiology of the disease had been derived from outbreak-
associated cases. Information on whether the majority of these sporadic cases
result from foodborne transmission was being provided by ongoing and active
disease surveillance. For instance, CDC had conducted a population-based active
surveillance for L. monocytogenes infections. Case- patients were identified and
controls were selected and matched on age, geographic location, socioeconomic
status, and underlying health conditions (Barnes et al., 1989).
Reported outbreak-associated listeriosis cases represent a small proportion of the

annual number of listeriosis cases estimated to occur in the U.S. (Mead et al., 1999).

For the period 1970 through 2000, 50 published outbreak investigation reports and
two unpublished investigation reports were reviewed (FDA et al., 2001). Data from
outbreaks from within and outside the U.S. were collectively summed by number of
outbreaks and number of cases and each food group was ranked accordingly. When
ranked by number of associated outbreaks, dairy products ranked number one,
followed by meat products, fish products and finally, vegetables. When numbers
of outbreak-associated cases were ranked, meat products were first, dairy products
were second, followed by vegetables and finally, fish. Serotype 4b was found in 16
(72.7%) of 22 outbreaks and serotype 1/2a was found in four (18.0%) outbreaks (FDA
et al., 2001).
2.7 Diseases Associated with L. monocytogenes

In 1929, the first case of human listeriosis was described (Nyfeldt, 1929). Since
then, listeriosis has been recognised as a rare but often fatal illness. Two high risk
(susceptible) subpopulations to listeriosis were elderly and perineonatal. In adults,
the disease listeriosis can have a long period of incubation time of up to three months
(Gellin & Broome, 1989) and it is characterised by the onset of severe symptoms
including meningitis, septicemia, primary bacteremia, sepsis, endocarditis,
nonmeningitic central nervous system infection, and flu-like illness including
fever, fatigue, malaise, nausea, cramps, vomiting, and diarrhea (Donnelly, 1994).
Nevertheless, meningitis and sepsis are the most commonly recognised symptoms
when susceptible adults contract the disease. Of 641 human cases, 73% of victims
had meningitis, meningoencephalitis, or encephalitis (Nieman & Lorber, 1980).
However, gastroenteritis illness (listerial gastroenteritis) from L. monocytogenes

has only recently been recognised as a distinct entity (Dalton et al., 1997). There
are two types of diseases associated with L. monocytogenes; invasive and non-
invasive. The invasive disease normally occurs in people with weakened immune
system. Those at risk include pregnant women and their foetuses, new born children,
the elderly and those who are immunosuppressed by medication (coricosteroids,
cytotoxic drugs, AIDS patients), especially after organ transplantation or illness
(haematologic malignancies such as leukaemia, lymphoma, and myeloma as well as
solid malignancies). The non-invasive disease can occur in anyone that has consumed
a high number of L. monocytogenes cells (Rocourt & Cossart,1997).
Typical signs and symptoms associated with the mild form of L. monocytogenes
infection are primarily those associated with gastrointestinal illness: chills, diarrhoea,

headache, abdominal pain and cramps, nausea, vomiting, fatigue, and myalgia. A
variety of foods have been implicated as the vehicle of infection. The frequency of
these types of outbreaks is unknown because most cases of mild listerial gastroenteritis
are not reported to public health officials (Centre for Disease Control and Prevention,
1999).
It is not known why the incubation period of listeriosis varies so much (more
than 24 hours to 91 days). It is possible that the dose of organism ingested, the host
immune system, and any intercurrent viral or bacterial infection play some role in
determining the period of onset of the illness after initial exposure to the organism
(Farber & Peterkin, 2000). The infective dose of L. monocytogenes depends on many
factors, including the immunological status of the host, microbial characteristics
of the strain involved and the particular type of contaminated with Listeria. The
occurrences and the course of infection may well depend on virulence factors and
infective dose (Rocourt & Cossart, 1997). The differential diagnosis and treatment of
L. monocytogenes infections is summarised in Table 4.
Table 4: Differential diagnosis and treatment of L. monocytogenes infections

(Armstrong et al., 1994).
Category Differential Diagnosis Treatment

Pregnancy infections - Influenza Ampicillin or penicillin
- Pyelonephritis
- Septic abortion
Granulomatosis - Neonatal sepsis Ampicillin and gentamicin
infantiseptica - Neonatal meningitis due to
enteric bacilli or group B
Streptococcus
Sepsis - Sepsis of unknown source Ampicillin or penicillin
due to a large variety of
organisms
Meningoencephalitis - Psychiatric illness or Ampicillin or penicillin,
metabolic encephalopathy possibly with gentamicin
- Infection due to intrathecally
Streptococcus pneumoniae,
Haemophilus influenza or
Neisseria meningitides in
immunosuppressed patients
Cerebritis - Brain abscess, tumor, stroke Ampicillin or penicillin
Focal infections - Varies with site Ampicillin or penicillin

CHAPTER 3
FACTORS AFFECTING GROWTH AND SURVIVAL OF

Listeria sp. IN FOODS
3.1 Temperature
The temperature range that support the growth of L. monocytogenes is in the range
of –2°C to 45°C (ICMSF, 1996). This bacterium can withstand freezing (El-Kest
& Marth, 1989), frozen storage (Hof et al., 1986) and thawing (Bryan, 1969). The
mean of minimum growth temperature on trypticase soy agar (TSA) of 78 strains of
L. monocytogenes was found to be 1.1°C ± 0.3°C, with a range of 0.5 to 3.0°C
(Juntilla et al., 1988). Two strains of Listeria sp. were able to grow at 0.5°C, and
eight strains of Listeria sp. grew at or below 0.8°C in 10 days as determined
with a plate-type continuous temperature gradient incubator. With 22 other strains
(19 strains of L. innocua and 1 each of L. welshimeri, L. grayi, and L. murrayi), the
minimum growth temperature ranged from 1.7°C to 3.0°C, with a mean of 1.7°C
± 0.5°C (Junttila et al., 1988). It was also found that haemolytic strains of Listeria
grew better than non-haemolytic strains under cold conditions.
A study was carried out by Rosenow and Marth, 1987 in order to determine the
generation times of L. monocytogenes at 35°C, 13°C, and 4°C in autoclaved skim
milk, whole milk, chocolate milk, and whipping cream. At 35°C, the generation
times of L. monocytogenes are 0.65 h in chocolate milk, 0.67 h in cream,
and 0.69 h in skim milk and whole milk. At 13°C, the generation times are 5.0 h in
chocolate milk, 5.3 h in cream, 5.9 h in whole milk, and 7.2 h in skim milk. At 4°C,
generation times are 1.27 to 1.56 days in skim milk, 1.26 to 1.50 days in whole
milk, 1.34 to 1.52 days in chocolate milk, and 1.16 to 1.66 days in cream. It was
found that the addition of cocoa powder, cane sugar, and carrageenan enhances the
growth of L. monocytogenes in milk (Rosenow & Marth, 1987). In other study, three
strains of L. monocytogenes exhibited generation times of 62 to 131 hours in chicken
broth and pasteurised milk, respectively, during extended incubation at -0.1°C to
–0.4°C (Walker et al., 1990).
Growth temperature may also influence the virulence of L. monocytogenes.

Growth at 4°C significantly increased the virulence of three clinical listerial

isolates in mice when compared to cells grown at 37°C for intravenously, but not
intragastrically, inoculated mice. It was also shown that growth at 4°C significantly
decreased the killing of test strains by human neutrophils. The virulence of L.
monocytogenes strain 10403S did not increase when grown at low temperature or in
increased levels of NaCl (Wood & Woodbine, 1979).
3.2 pH
L. monocytogenes can only grow at pH values from 4.1 to 9.6, with optimal growth at
neutral pH (Petran & Zottola, 1989). The ability of 16 strains of L. monocytogenes to
grow in a nutrient medium acidified with HCl to pH values between 4.2 and 7.0 has
been studied (George et al., 1988). Growth of L. monocytogenes in culture media
has been observed at pH 4.4 in less than 7 days at 30°C and 14 days at 20°C. The
growth of L. monocytogenes occurred at a pH less than 5.0 when grow at 10°C and
7°C, whereas at 4°C, growth of this bacterium occurred at pH 5.23. The minimum
pH values of this bacterium at its corresponding temperature were at 30°C was 4.39,
20°C was 4.39, 10°C was 4.62, 7°C was 4.81 and 4°C was 5.23 (George et al., 1988).
Principally, when pH was lowered to 5.0, the survival time of L. monocytogenes

was significantly reduced. However, Listeria would grow at pH 5.0 at temperatures
greater than 13°C. The minimum growth pH of a bacterium is influenced by many
factors, such as temperature of incubation, general nutrient composition of growth
substrate, water activity (aw), and the presence and quantity of NaCl and other salts
or inhibitors (Jay, 1996).
3.3 Acid
Organic acids are generally more effective in eliminating microorganisms than
inorganic acids due to their lipophilic nature (Corlett & Brown, 1980). Organic acids
accentuate the pH inhibition of growth rates of this bacterium and limits the magnitude
of that inhibition to the concentration of the undissociated acid (Presser et al., 1998).
Based on the degree of undissociation, acetic acid was the most detrimental to L.
monocytogenes, followed by lactic acid and citric acids. The growth-inhibitory pH
was found to be 5.0 for propionic acid, 4.5 for acetic and lactic acids, and 4.0 for
citric and hydrochloric acids at 4°C. Based on many studies, it was clear that pH,
acid type and temperature influence the growth and survival of L. monocytogenes
(Ahamad & Marth, 1989; Farber et al., 1988).

Factors Affecting Growth and Survival of Listeria sp. in Foods / 17
3.4 Salt
L. monocytogenes is tolerant to NaCl where it was capable to grow in 25.5% and
survived for one year in 16% NaCl (Stenberg & Hammainen, 1955). Survival times of
this bacterium in media containing 25.5% NaCl increased from 3 days at 37°C to 24
days at 22°C (Shahamat et al., 1980). As with pH and temperature, the effect of salt
on microorganisms is often examined with other parameters. Thermal inactivation
of L. monocytogenes was not significantly influenced by the presence of up to
2% NaCl, but heat-stressed cells of L. monocytogenes had increased sensitivity
to the salt. The effect of salt concentrations (4-6%) on L. monocytogenes can be
summarised that low concentration of salt (4-6%) could improve the survival of this
bacterium, while higher concentrations (more than 10%) may reduce the survival of
Listeria cells at limiting pH values (Martin & Fisher, 2000).
3.5 Water Activity

The effect of water activity on the growth and survival of L. monocytogenes was studied
using brain heart infusion broth, three humectants, and a t 30°C incubation.
Results showed that the minimum aw that permitted growth of serotypes 1, 3a, and
4b of L. monocytogenes were as follows: 0.90 with glycerol, 0.93 with sucrose, and
0.92 with NaCl (Farber et al., 1992). In different study using trypticase soy broth
at a pH of 6.8 and incubation at 30°C, the minimum aw that permitted growth of
L. monocytogenes was 0.92 with sucrose as humectant (Petran & Zottola, 1989). In
view of these findings, L. monocytogenes is the second only to the Staphylococcus as
a foodborne pathogen that able to grow at aw values less than 0.93.
3.6 Modified Atmosphere

The growth of L. monocytogenes is reported to be little affected by anaerobic, or
oxygen reduced atmosphere (ICMSF, 1996). However, growth of this bacterium is
reduced by carbon dioxide as commonly used in modified atmosphere packaging
(MAP). In fresh trout, MAP with 60% CO2: 40% N2 or 40% CO2: 30% N2: 30%
O2 prevented growth at 5°C for more than two weeks of storage although under
aerobic storage growth was detected after 4 days (Davies, 1997). In smoked blue
cod, growth of L. monocytogenes observed at –1.5°C under vacuum packaging was
prevented after 160 days under MAP with 100% with carbon dioxide.
The MAP packaging system may reduce the growth rate of this bacterium and
the yield substantially when incubated at 3°C (Bell et al., 1995). Similarly, Ingham
et al., (1990) reported the inhibition of growth by high level of carbon dioxide,

but only at low temperatures. Szabo & Cahill (1998) had studied the combined effect
of storage atmosphere, temperature and nisin on the growth of L. monocytogenes.
At 4°C and with 100% of carbon dioxide, growth rate in buffered tryptone soy
broth was reduced, and lag times increased. However, the addition of bacteriocin
ALTA 2341 was required to prevent growth of this bacterium for at least 21 days. In
addition, strictly anaerobic conditions significantly increased the recovery of heat-
injured Listeria when compared with aerobically controls (Knabel et al., 1990). The
summary of growth limits of L. monocytogenes is shown in Table 5.
Table 5: Limiting conditions for growth of L. monocytogenes

(Ryser and Marth (1991) and ICMSF (1996)
Environmental factor Lower limit Upper limit

Temperature (°C) -2°C to +4°C ~ 45°C
Salt (% water phase NaCl) 13 to 16% 25.5%
aw (corresponding to salt %) 0.91 to 0.93 (>0.997)
pH (HCl as acidulant) 4.2 to 4.3 9.4 to 9.5
Lactic acid (water phase) 0 3.8 to 4.6 mM
MIC of undissociated acid (sodium lactate) 800 mM 1000 mM

CHAPTER 4
THERMAL INACTIVATION OF L. monocytogenes
4.1 Background of Thermal Inactivation

Historically, conflicting results have appeared in the scientific literature concerning
the thermal resistance of L. monocytogenes. Since 1985, a large number of studies
have been reported on its thermal destruction in dairy products. D-values have been
determined on many strains of L. monocytogenes in whole and skim milk, cream, ice
cream, and various meat products. Heat resistance appears to be dependent on the
inoculum level and the methodology used in each study.
4.2 Theory Death of Microbes by Heating

Heat treatment has been long regarded as one of the most widely used and most
effective means for destruction of spoilage and pathogenic microorganisms in
foods (Linton et al., 1995). The efficacy of thermal processes traditionally has been
calculated on the basis of the assumption that microbial mortality is a process
following a first-order kinetics (Ball & Olson, 1957; Stumbo, 1973; & Teixeira, 1982).
It means that, at a constant temperature, thermal death of bacteria is exponential
with time (Rahn, 1945).
The heat resistance of vegetative bacteria and microbial spores at a constant

temperature is characterised by their D-value; this is time required to reduce 90%
or one log cycle. For vegetative organisms, D-values are quoted in the range of 60-
80°C, and for spores in the range of 100-140°C. The number of decimal reductions,
log (N0 / N), can be evaluated from:
log10 (N0 / N) = - heating time/D (Equation 1)
where N0 is initial population and N is the final population (Peleg & Penchina, 2000).
Two important points from this equation: firstly, it is not possible to achieve 100%
reduction of microorganism; and secondly, for a specified heat treatment, the final
population will increase as the initial population increases. As in thermal processes,
the efficacy of the different methods, or of treatments under different conditions,
notably the pH and temperature, has been reported frequently and compared in term
of the D-values that they produced (Peleg &Penchina, 2000).

According to Casolari (1988), the concept of a D-value becomes problematic

when the experimentally determined semilogarithmic survival curves are clearly
non-linear. Indeed, numerous examples of non-linear survival curves for bacteria
exposed to heat have been reported in the literature (Pflug & Holcomb, 1983;
Tomlins & Ordal, 1976). These studies have shown that most survival curves display
an initial “shoulder” followed by exponential decline. Many curves also contain a
“tailing” region. Several authors claimed that, the different shapes of the survival
curves are a manifestation of the underlying distribution properties, namely, mean
or mode, standard deviation, skewness, and others. Shape changes that occur by
changing the external conditions (temperature, agent concentration, pH or others)
are therefore interpreted as reflecting changes in the distribution parameters, like a
shift of the mode, an increase or decrease of the standard deviation and others (Peleg
et al., 1997).
Various ways has been used in resolving the difficulty in determining D-values for
non-linear curve. In many cases, the curvature has been simply ignored and a straight
line was forced on the data. The slope of this line was treated as a rate constant from
which a D value was extracted. This is a rather crude and arbitrary procedure and
can result in over- or under processing, depending on whether the semilogarithmic
survival curves have upward or downward concavity, respectively. Another solution is
to divide the survival curves to several segments, each small enough to be estimated
by a straight line. However, because there are no firm guidelines on how to select the
segments, such an approach still leaves an arbitrary element and can considerably
increase the complexity of the calculation procedure. According to Stumbo (1973)
and others (Körmendy & Körmendy 1997), a semilogarithmic survival curve is or
can be evidence that the population is a mixture of subpopulations each having a
mortality pattern that following a first-order mortality kinetics. However, because it is
difficult to explain every departure from linearity as evidence of the creation of a
new mixture, several authors have developed more elaborate population balance
and kinetics models that are consistent with the actual shapes of the survival curves
(Sapru et al., 1992; Sapru et al., 1993; Whiting, 1995; Körmendy and Körmendy,
1997; Gerwen & Zwietering, 1998).
Another analytical method for the thermal inactivation of microbes is based on

chemical reaction kinetics (Bailey & Ollis, 1986). The microorganisms are considered
to be inactivated by a first-order reaction at a constant temperature, which is similar
to the inactivation of biochemical substances in food (Bigelow, 1921). The rate

Thermal Inactivation of L. monocytogenes / 21
constant of inactivation, k in the Arrhenius model corresponds to the D-value in

the z-value model. D-value and z-value are two parameters that have been used to
estimate thermal inactivation of microorganisms during heating process. z-value is
obtained as the reciprocal slope from log D-value versus temperature, which also
demonstrates linear relationship. The D-value is equal to 2.303/k. The temperature
used in the z-value model is the reciprocal of the temperature used in the Arrhenius
model (Fujikawa & Itoh, 1998).
According to Busta (1978), the existence of biphasic curve is often observed as the
microorganisms are exposed to environmental stress. It is generally associated with
heterogeneous cell subpopulation in their heating menstruum, which corresponds
to dual thermal inactivation mechanisms. If the survival curve is biphasic curve, it
may contain growing cells, and their bacterial growth rate based on generation time.
Traditionally, the lag time and exponential rate of growth have been determined by
drawing a line through the exponential phase of the curve or by drawing a tangent
to the steepest part of it. The time taken for a doubling of the population (generation
time), can be determined from the slope of this line. When log10 (cell density) is
plotted against time, the relationship between slope and generation time is:
log10 2
slope of steepest tangent = ---------------------- (Equation 2)
to the exponential growth phase generation time
A straight line result as the log10 number of cells is plotted against time in a
semilogarithmic graph. The straight line function is an immediate indicator that the
cells are growing exponentially. The semilogarithmic graph is convenient and simple
to use for calculating generation time from a set of results. The doubling time may be
read directly from a semilogarithmic graph (McMeekin et al., 1993).
Interpretation of these kinetic models involves predictive microbiology, which

relies upon the application of mathematical modeling techniques and equations
to predict bacterial rate constant. The prerequisite to the development of reliable
mathematical models is the collection and appropriate processing of good quality data
describing the effects of different factors on microbial development. The successful
implementation of predictive microbiology requires appropriate technology to be
developed. This will almost certainly take the form of interactive computer software
based on mathematical models summarising large databases of growth responses,
and may involve the development of smart sensors which record and interpret the

effect of environmental parameters on the growth of microorganisms of interest in a

product or process (McMeekin et al., 1993).
The recent realisation of the importance of foodborne transmission of L.

monocytogenes in regard to the etiology of both epidemic and sporadic outbreaks
of listeriosis has led to a need for data on how its growth characteristics are affected
by various food formulation and storage factors. Buchanan & Philips, (1990) have
summarised the comparison of selected reported growth kinetics for L. monocytogenes
versus those predicted by the cubic model.
4.3 Heat Resistance of L. monocytogenes in Foods

Generally, heat resistance of L. monocytogenes is appreciably more resistant than
common Salmonella serotypes and other non-sporing foodborne pathogens, but less
resistant than Salmonella senftenberg 775W (Mackey & Bratchell, 1989). The authors
summarised the most of D-values (decimal reduction time) which are those quoted
from scientific literature and based on detailed kinetic data, and despite differences
in heating media, methods, and test strains, the equation was derived from a straight
line plot of log D-values versus temperature. The equation is:
log D = 10.888 – 0.141519 t
where D is the decimal reduction time in s and t is the temperature (°C), the z-value
is 6.9°C (S.E. : 0.136). Many separate estimates of z-values have been published,
ranging from 4.3 to 9.9. The mean of 27 of such estimates was 6.7. These consensus
values for L. monocytogenes are higher than usual for vegetative cells, which typically
have z-values around 5.0 in media of higher water activity (Tomlins & Ordal, 1976).
4.3.1 Dairy Products
A summary of thermal D-values and z-values for some L. monocytogenes

strains was reported by Jay (2000). The D-values indicate that the high-
temperature, short-time (HTST) protocol for milk (71.7°C for 15 seconds)
is adequate to reduce normally existing numbers of this organism below
detectable levels. The vat or low-temperature, long time (LTLT) pasteurisation
protocol (62.8°C for 30 minutes) is even more destructive. Employing the
Scott A strain (serovar 4b from the Massachusettes outbreak), D-values
ranged from 0.9 to 2.0 seconds under HTST pasteurisation with z-values of
6.0 to 6.5°C. The F5069 strain (serovar 4b) appeared to be a bit more heat

resistant than Scott A from these results, although Scott A was the most heat
resistant of three other strains evaluated, not including F5069 (Bradshaw et
al., 1985).
The thermal resistance of L. monocytogenes is not affected by the intracellular

position. With Scott A freely suspended in whole raw milk at mean level
of 2.6 X 105 CFU/ml and heating at 71.7°C for 15 seconds, no survivors
could be found after five heating trials (Lovett et al., 1990). In seven heating
trials with Scott A engulfed in vitro by bovine phagocytes, no survivors
could be detected with a mean number of log10 4.70 CFU/ml. Further, these
investigators experimentally infected cows with Scott A and were still unable
to find survivors following 11 pasteurisation trials at 71.7°C for 15 seconds
with numbers of Scott A that ranged from 3.15 log10 CFU/ml to 4.00 log10
CFU/ml (Malinverni et al., 1985).
Employing five strains of L. monocytogenes (included serotypes 1,3 and 4)

in whole milk, skim milk, and 11% non-fat milk solids, Donnelly and Briggs
(1986) found that composition did not affect heat destruction and that at
62.7°C, the D-values were 60 seconds or less. When milk that was naturally
contaminated with a serotype 1 strain at around 4.00 log10 CFU/ml was
subjected to an HTST protocol at temperature ranging from 60°C to 78°C, no
viable cells could be detected at processing temperatures of 69°C or above
(Farber et al., 1988). In addition, Mackey & Bratchell (1989) concluded that
normal pasteurisation procedures will inactivate this organism but that the
margin of safety is greater for the vat protocol (LTLT) than the HTST protocol.
Their mathematical model predicted a 39 D for vat and a 5.2 D for HTST.
4.3.2 Red Meat and Poultry Products
There are several reports that are unusually resistant when heated in
meat or poultry products but few quantitative data are available. In 1980,
Karaioannoglou and Xenos isolated the viable organisms from grilled
meatballs cooked to an internal temperature of 78°C to 85°C, when initial
inoculum was 103/g, but not when it was 102/g. Apparently exceptional
resistance was also detected ‘large numbers’ of survivors in chicken breasts
cooked to an internal temperature of 65.5°C to 71°C, whilst smaller numbers
were recovered even after cooking to 82°C. The Agriculture Department of
the United States has issued a statement that cooking hot dogs to 71.7°C is
‘borderline’ for the destruction of Listeria; an internal of 71.7°C resulted in

a 3-D reduction, whilst 68.3°C resulted in a 2-D reduction (Anon, 1988).

Regrettably, no details of holding times were provided.
Others have not observed unusual resistance in meat and poultry products.
Careful studies by Lund et al., (1989) showed that microwave cooking of
poultry to an internal temperature of approximately 70°C resulted in a
106/g fold lethality consistent with that predicted from detailed kinetics data
available from the milk studies. The D-values at 60°C, 65°C and 70°C were
approximately 4.5, 0.65 and 0.15 min, somewhat higher than milk, but
consistent with data for heating in broth (D60 was between 3.6 to 5.4 min)
(Lund et al.,1989).
Most reports of unusual resistance derived from trials with cooked foods. It
seems likely that uneven heating accounts for the apparent thermoresistance
in these cases. However, the possibility of protection by fat or drying during
heating must also be considered, and good data are lacking on this. The
addition of 30% fat to beef enhanced survival of L. monocytogenes, but the
D-values at 62.8°C in the presence of fat (1.5 min) was only about twice
that in milk at the same temperature (Anon, 1988). A greater increase in
resistance occurred on addition of salt, spices and curing salts to sausage
meat (Farber et al., 1988). D-values at 60°C and 62°C (calculated from
reported data) increased 4- to 5-fold from 2.7 and 0.4 min respectively in
the absence of additives to 15 and 1.7 min in their presence.
4.3.3 Other Foods
L. monocytogenes in liquid whole egg was slightly more resistant than in

milk (D-values at 62°C were 72 and 108 s compared to 62 s in milk) and
unusually large z-values (9.0, 9.9) were reported (Leasor & Foegeding, 1988).
Resistance in cabbage juice was appreciably less than in milk with D56 of
2.04 and 3.64 min at pH 4.6 compared to 9.9 min in milk. The acid pH of
cottage cheese (pH 5.0-5.4) did not ensure destruction of L. monocytogenes
during cooking of curds containing 105-106 organism/g, although the
numbers surviving the heat treatment (57.2°C for 30 min) were very
low and could be detected only by enrichment (Ryser et al., 1985). Spray
drying of dry milk also does not ensure destruction of L. monocytogenes;
with inlet and outlet temperatures of 165°C and 67°C, there was only a
10- to 30-fold reduction in numbers (Doyle, 1999). The heat resistance in
autoclaved carrot was similar to that in chicken and meat (Gaze et al., 1989).

The heat resistance of L. monocytogenes in seafood products is summarised

in Table 6 (Anon, 2001).
Table 6: Heat resistance of L. monocytogenes in seafood products (Anon, 2001)

Temperature D-value1 Medium
°C °F (min)
50 122 34.48 Blue crabmeat
51.6 125 97.0 Lobster
54.4 130 55.0 Lobster
55 131 10.23 Crawfish tail meat
56 132.8 48.09 Mussels, brine soaked
57.2 135 8.3 Lobster meat
58 136.4 10.73 Salmon
58 136.4 7.28 Cod
60 140 2.39 Lobster meat
60 140 5.49 Mussels, brine soaked
60 140 4.48 Salmon
60 140 1.98 Cod
62 143.6 2.07 Salmon
62 143.6 0.87 Cod
62.7 145 1.06 Lobster meat
65 149 0.87 Salmon
65 149 0.28 Cod
68 154.4 0.15 Salmon
68 154.4 0.15 Cod
70 158 0.07 Salmon
70 158 0.03 Cod
1D-value (decimal reduction value) is the time required to kill 90% of a bacterial population at
a specific temperature in a specific medium
4.4 Effect of Sublethal Heating on Thermotolerance

It has been demonstrated that when Salmonella spp. cells grown at 35°C are held
at 42-48°C before being heated at higher temperatures, the heat resistance of these
so-called ‘heat-shocked’ cells will increase (Mackey & Derrick, 1987). Numerous
studies have been done to observe if a similar phenomenon could be demonstrated
with a Gram-positive organism like L. monocytogenes. The results concerning heat
shocking of L. monocytogenes are conflicting, with some investigators reported
no effect (Bradshaw et al., 1985; Bunning et al., 1990), whereas others reported
increased resistance (Farber & Brown, 1990; Fedio & Jackson, 1989; Linton et al.,
1990).
Thus, experiments were carried out on inoculated meats, where inoculated meat
bags were held at 48°C for 30 min, before being exposed to higher test temperatures.

The results of one series of experiments are shown in Table 7. Heat shocking of
L. monocytogenes in some cases, resulted in an almost 2-log increase in survival as
compared to cells which had not been heat shocked. In one study, the heat shocking
of strain Scott A at 48°C for 20 minutes resulted in a 2.3-fold increase in D-values at
55°C (Linton et al., 1990). In another study employing Scott A in broth and ultrahigh
temperature (UHT)-treated milk, an increase in heat resistance was observed
following exposure to 48°C for 60 minutes and subsequent exposure to 60°C.
Finally, in a study employing 10 strains at a level of about 107/g in a sausage mix

and heat shocking at 48°C for 30 and 60 minutes, no significant increase in
thermotolerance was observed at 62°C or 64°C, but those shocked for 120 minutes
did show an average 2.4-fold increase in D-values at 64°C (Farber & Brown,
1990). In this study, the thermotolerance was maintained for at least 24 hours
when the cells were stored at 4°C. This ‘heat-shock’ effect could be significant for
bulk food, which are heated slowly or other foods which receive a marginal heat
treatment. However, if sublethal heating does lead to greater thermotolerance, it
would not pose a problem for milk that contains fewer than 10 cells/mL assuming
that a two-fold to three-fold increase in D-values occurs (Jay, 1996).
Table 7: The effect of prior heat shock on the heat resistance of L. monocytogenes in
cured meat at 64°C (Farber & Brown, 1990).
Time (min) of heating Heat shock1 Viable Listeria in cured
meat (log10 CFU/g)
0 + 6.2
- 4.8
2 + 5.5
- 3.9
4 + 4.9
- 3.1
6 + 4.2
- 2.4
8 + 3.7
- 2.3
1
Cells were heat-shocked at 48°C for 30 minutes before being heated at the test temperature of
64°C
4.5 Sublethal Injury at High and Low Temperature Stress of L. monocytogenes

A variety of environmental changes from normal conditions for microorganisms are

stressful and may inflict injury to the organism. The multiple process stages used
in foodservice systems and food processing plants involve the use of elevated or
reduced temperatures that can result in sublethal injury to bacteria (Sawyer & Pestka,
1985). Sublethal injury of bacteria can be defined as the inability of the bacteria to
multiply in a medium that contains a selective agent that has no inhibitory action on
unstressed cells (Busta, 1976). It is a temporary loss of tolerance for specific conditions
in a microorganism (Sørhaug, 2000). Injury may result from many food processing
and handling methods, including thermal treatment, refrigeration, freezing, drying,
and irradiation, or from exposure to preservatives, acidity, or low water activity.
Equipment surfaces that have been sanitized, as well as used in processing of foods or
cleaning of equipment, may contain injured microorganisms. Available data indicate
that both Gram-negative and Gram-positive bacterial cells, bacterial endospores,
yeast, and molds may be injured by stresses (Adams, 1978; Busta et al., 1981; Ray,
1979; Tracy & Duncan, 1974; Foegeding & Ray, 1992).
Research has indicated that, for injured vegetative cells, regardless of the nature
of stress imposed on a microbial population, (a) the injury is repaired when
the injured organisms are held in an appropriate environment, (b) the optimum
temperature and time required for repair may vary with the nature of stress, (c) the
completely repaired cells regain their normal resistance to the selective agents in
the media, and (d) the process of repair must precede cell multiplication (Flowers &
Ordal, 1979; Hartman, 1979; Ray, 1979). Therefore, it is desirable to allow stressed
cells to repair any damage before they are isolated or enumerated by customary
procedures on selective media. The procedure that promotes maximum repair must
be evaluated to eliminate or minimise multiplication of competing organisms. In other
words, characteristics of injured bacteria are as follows; (i) enhanced sensitivity
to surface-active compounds, organic acids, salts, dyes, and selective media; (ii)
loss of cellular materials, (ii) increased lag time; and (iv) inability to multiply until an
adequate resuscitation period has occurred (Jay, 2000).
Selective compounds such as surface-active agents, salts, antibiotics,

sulfanilamides, acids, and dyes are added to media for the selective and differential
detection of indicator, pathogenic, spoilage, or other microorganisms from foods.
Some of these agents are inhibitory to the process of repair while others are toxic and
cause death of the injured organisms. This suggests that when such media must be
used, the injured microorganisms in the samples must be permitted to resuscitate
in a suitable environment prior to their exposure to selective media (Flowers & Ordal,

1979; Hartman, 1979; Ray, 1979). The use of selective media without appropriate
precautions to permit regular repair of injury may result in failure to detect the
injured, but viable and potentially normal microorganisms. Hence, selective media
may underestimate the microbial content of particular food product.
Differential plate-count procedures (TSA with or without 4% NaCl) to reveal

that cells have been injured actually introduce additional or secondary stresses so
that cells that are reversibly injured in the first place (primary stress) do not repair
and reproduce. Because reversible injury and the lack of ability to form colonies
have often been related to microbiological analysis and the use of selective
media, differential conditions (secondary stresses) regularly seen are increased salt
concentrations, deoxycholate, lauryl sulfate, bile salts, detergents, crystal violet,
brilliant green, and other dyes, azide and antibiotics. Secondary stress may also
occur during the dilution of samples, thus Mg2+ containing diluents are recommended
for recovery of stressed cells. Similarly a cold shock during dilution may kill some
bacteria. The mechanisms of injury vary in vegetative cells and spore (Sørhaug, 2000).
Basic investigations to determine the sites of cellular damage and mechanism of

cellular repair have been reported (Ray, 1986). From these results, it become apparent
that irrespective of the differences in sublethal treatments (such as freezing, drying,
and heating) and bacterial species, injured cells had considerable similarities in
manifestations of injury. Loss of UV-absorbing cellular materials to the environment
and sensitivity too many selective compounds were common in most treatments
(Hurst, 1977; McLeod & Calcott, 1976; Ray & Speck, 1973; Ray et al., 1971). Some
differences in the mechanisms of repair (Hoover & Gray, 1977; Ray & Speck, 1973;
Sinskey and Silverman, 1970) were observed between laboratories, probably due
to the plating media containing different selective agents to measure injury (such
as NaCl, bile salts, deoxycholate and others) and the use of different metabolic
inhibitors to study repair processes. As more data became available, similarities in
the mechanisms of injury repair incurred by different sublethal treatments became
evident. Injury in certain cellular structure resulted from conformational alteration
of macromolecules present in these structures. Repair of this injury involved
reorganisation of macromolecules to their original conformation. Some of this repair
could not be detected by methods used to study de novo synthesis as described by
Ray (1986).
The cell envelopes of Gram negative bacteria are much more complex than
those of Gram positive organisms. Gram positive bacteria cell envelopes consist of
a cytoplasmic membrane made of phospholipid and protein, surrounded by a cell

wall composed largely of peptidoglycans and teichoic acids and/or polymers. The
Gram-negative bacteria envelope has two membranes, the cytoplasmic (inner) and
the outer one. Both contain phospholipid plus protein, and between them lies a
layer of peptidoglycans linked to the outer membrane by lipoprotein. In addition,
the outer membrane of Gram-negative bacteria contains lipopolysaccharides, the
hydrophilic polysaccharide portions of which protrude from the outer membrane
and form the somatic ‘O’ antigens. Various enzymes are located in the area between
the two membranes (the ‘periplasmic space’) as well as in the membranes of both
Gram-positive and Gram-negative bacteria. Either type of bacterium may possess
capsules made of protein or polysaccharide (Mossel et al., 1995).
The outer membrane of Gram negative bacteria is believed to act as a physical

barrier that protects the enzymes present in the periplasmic space and those
associated with the inner membrane against inhibitory substances. Fully viable Gram
negative bacteria area, consequently, resistant to higher concentrations of a number
of membrane-active antimicrobial substances that affects Gram-positive bacteria at
lower levels. These include bile salts, and triphenylmethane dyes, such as crystal
violet and brilliant green, and cationic detergents (Maxcy, 1970; Scheusner et al.,
1971; Roth et al., 1973). Damage to the outer membrane incurred by exposure to
a variety of treatments or agents, such as heating, drying, freezing, chilling, low
pH, detergents and disinfectants (Mackey, 1983), allows substances that negatively
affect microbial growth to permeate and this reduce the resistance of Gram-negative
organisms such as E. coli and Salmonella spp. to these inhibitory agents.
Similar phenomena occur in Gram positive non-sporing bacteria, such as

L. monocytogenes as a result of sublethal injury. Damage to the cell wall and/
or cytoplasmic membranes make injured S. aureus sensitive to NaCl, and causes
Enterococcus faecalis to be inhibited by concentrations of NaCl and potassium
tellurite that are well tolerated by fully viable populations. Moreover, damage to cell
membranes in both Gram-positive and Gram- negative bacteria results in the loss
(’leaching’) of cell components, particularly amino acids, nucleic acids, citric acid
cycle components and mono- and divalent cations, negatively affecting the growth.
Apart from membranes and cell walls, other cell components are also commonly
affected by stress incurred due to exposure to adverse physical and chemical
factors. These include: (i) DNA, sensitive to ultraviolet and γ-radiation, heating and
some antibiotics; (ii) RNA, susceptible to heating, freezing and drying, and a number
of antibiotics; and (iii) cell proteins and enzymes. Some of these effects may be

linked with cytoplasmic membrane damage, since many components (e.g. enzymes,
nucleic acids) are associated with the membrane (Mossel et al., 1995).
4.6 Recovery of Sublethal Injury of L. monocytogenes

Injury of microorganisms implies a temporary condition. If it were not temporary, the
permanent change would be interpreted as a mutation and therefore could not be
defined as injury. The cells are classified as injured rather than inactivated or dead
when they have the capability to function in an unrestrictive environment and can
regain a normal physiological state concomitant with initiation of growth and cell
division. This restoration of the capabilities lost in damage from the environmental
stress has been defined or identified as a recovery or repair process. It also has been
termed resuscitation, implying that the cells have been revived from apparent death
(Busta, 1976; Busta, 1978).
Investigation of specific nature of sublethal damage in some structural and

functional macromolecules of bacterial cells was very useful in developing several
resuscitation method for recovering indicator and pathogenic bacteria from preserved
foods (Ray, 1986). In general, several concepts were used to develop these procedures:
(i) injured cells could lose culturability if exposed to a selective enrichment; (ii) most
injured cells repaired within 2 hours at a suitable incubation temperature
in a nutritionally-rich non-selective medium, and (iii) repaired cells regained the
characteristics of normal cells, including to multiply (Ray, 1986).
The value of repair period before selectively isolating Salmonella from dried egg
products was recognised by North (1961). He used lactose broth as a nonselective
pre-enrichment medium prior to selective enrichment in either selenite cystine or
tetrathionate broth. The success of this method was attributed to resuscitation of
cells injured by the egg drying process. However, incubation for 24 hours in lactose
broth could allow overgrowth by competing microorganisms, which are present in
far greater numbers than Salmonella in the products. Recognising this, other workers
suggested a shorter pre-enrichment period (2 to 7 hour) (D’Aoust, 1981). Currently
recommended procedures for the isolation of Salmonella from processed foods call
for pre-enrichment of the sample in a suitable broth for 24 hour at 30°C to 37°C
before proceeding with the selective enrichment (Foegeding & Ray, 1992).
Growth conditions that influence the composition of the cell or the physiological
state of the microorganism may affect its susceptibility to damage by subsequent
exposure to stress. These same factors may also influence the repair of microorganisms

after injury. As a consequence, these factors then influenced the growth of damaged
cells. The factors are specific nutrients, pH, temperature, gaseous atmosphere, culture
age, redox potential, osmolality, water activity, ionic strength, salts, surface tension,
and storage (Busta, 1978). In addition to these, the selective agents generally prevent
repair of injured cells leading to underestimation of potential disease producing
organisms. Smith & Archer (1988) found that the selective agents used in media
for the isolation of Listeria species such as 0.25% phenylethanol, 0.0012% acriflavin,
0.01% potassium tellurite, 0.01% polymyxin B sulfate, 5% NaCl or a combination
of these ingredients did not allow resuscitation of heat injured Scott A strain of L.
monocytogenes.
The accumulation of hydrogen peroxide appears to be a universal response in

cells undergoing injury and injured cells appear to have an increased sensitivity
to the toxic effects of hydrogen peroxide (Martin et al., 1976). Dallimier & Martin
(1988) have shown that the catalase activity of heated cell extracts of four strains
of L. monocytogenes decreased sharply at temperatures between 55°C and 60°C
and superoxide dismutase was even more heat labile than catalase. The addition of
catalase during the repair period generally is beneficial in removing toxic hydrogen
peroxide. However, adding catalase to either solid or liquid media is not a simple
matter since sterilisation of catalase is difficult. The non-enzymatic hydrogen
peroxide decomposer, sodium pyruvate, appears to be equally effective as catalase
in removing hydrogen peroxide and is much easier to add to media since it can be
autoclaved in situ. The addition of pyruvate to selective media enhanced the recovery
of injured microorganisms ( Hendry et al., 1976; Martin et al., 1976; Brewer et al.,
1977), however the addition of pyruvate is not effective in aiding resuscitation of heat-
injured L. monocytogenes for uncertain reason (Smith & Archer, 1988). Nevertheless,
Farber et al. (1988) indicated that addition of pyruvate (concentration not given)
to modified McBride agar enhanced recovery of heat injured L. monocytogenes. It
was necessary to incubate the plates at 22-25°C for 7 days in order to demonstrate
enhancement of recovery by pyruvate; however, no data were presented.
Mackey et al. (1994) examined the recovery of heat-injured L. monocytogenes

as a function of incubation temperature and composition of recovery medium. The
authors found that heat-injured cells exhibited a broad optimum temperature for
recovery centered around 20-25°C. The best recovery medium of those tested was
blood agar. Incubation of cells in broth or chicken slurry at 5°C (cold-enrichment)
did not allow repair of potentially lethal injury, as it did not allow recovery of cells

that would otherwise have died if incubated at a higher temperature. In some cases
incubation of heat-injured cells at 5°C resulted in death of a proportion of the
population. Repair of sublethal heat-injury, measured as the time of incubation in
tryptone soy broth needed to regain the ability to grow on Listeria selective agar,
was slower and less complete at 25°C than at 2°C; repair took 10-15 hour at 25°C
compared with 8-12 days at 5°C. Refrigeration of heat-injured foods should not
therefore increase the risk that heat-injured cells will recover from the heat treatment
(Mackey et al., 1994).
Recently, an effective solid medium for resuscitation of injured L. monocytogenes,

was developed and known as thin agar layer, TAL (Kang & Fung, 1999). Modified
Oxford Medium (MOX), a specific plating medium, inhibits heat-injured L.
monocytogenes from growing, whereas tryptic soy agar (TSA), a non-selective
medium, does not. In order to facilitate recovery of heat injured L. monocytogenes
cells while providing selectivity of isolation of L. monocytogenes from other bacteria
in the sample, a 14 ml of non-selective medium (TSA) is overlaid onto pre-poured
and solidified MOX medium. The injured L. monocytogenes repaired and started to
grow in the TSA during the first few hours after incubation of the plate. During the
resuscitation of injured cells, the selective agents from MOX diffused to the TSA top
layer to inhibit other microorganisms. A typical reaction (black colonies) shown by
L. monocytogenes on TAL after 24 hour of incubation at 37°C (Kang & Wu, 2001).

CHAPTER 5
STRESS AND EFFECTS ON VIRULENCE OF

L. monocytogenes
The term virulence can be defined as the degree of pathogenicity showed by a strain of
microorganism (Pelczar et al., 1993). This concept is intrinsically linked to the disease
and it is measured in term of morbidity and mortality. L. monocytogenes has its primary
priorities in life cycle; survival and multiplication (Mekalanos, 1992). The complex
interaction between survival and multiplication within host tissue and microbes
will affect the disease manifestation. The removal of one virulence factors from the
pathogen may or may not affect the ability of this microbe to cause illness (Madden,
1993). The understanding about the interaction between microbial virulence and
host defense mechanism will give clear meaning of disease manifestation (Madden,
1993). The microbial virulences have specialized mechanism of virulence factors
that are capable of unrestricted growth within these environment (Brubaker, 1985).
The virulence factors can be classified into extracellular parasites and intracellular
parasites (Brubaker, 1985). The pathogen restricted to extracellular location possess
determinants that permit resistance to phagocytosis. However, intracellular pathogens
may promote penetration of non-professional phagocytes or assure survival after
uptake by phagocytes, which involved in destruction of invading bacteria through
neutrophils, monocytes, and macrophages (Brubaker, 1985). These mechanisms are
important for the survival and multiplication of pathogen because it serve as primary
scavenger of invading bacteria, and it is the most effective non-specific mechanism
of host defense of microorganism.
L. monocytogenes is considered as important facultative intracellular parasites,

which able to multiply in the most of the environment exploited by extracellular
parasites. In addition, this bacteria is capable to penetrate and grow within a variety
of host cells (Brubaker, 1985). It also can exploit multiple in vivo environments and
thus become capable of causing some of severe diseases to human (Moulder, 1962).
Unlike typical extracellular parasites, facultative intracellular parasites generally
showed dependence on oxidative catabolic mechanisms that probably reflects the
elevated oxygen of host-cell cytoplasm (Brubaker, 1985). L. monocytogenes is also

capable to grow significantly in normal macrophages and some may even survive
residence within neutrophils and monocytes; where in most cases, L. monocytogenes
can also penetrate non-professional phagocytes. Although L. monocytogenes has
provided a definite model of understanding the nature of cellular immunity, the
nature of its virulence factors are not fully understood (Brubaker, 1985).
Recent research confirmed that the growth of this bacterium occurred equally
well within normal and peroxidase-positive macrophages and the factors that
stimulated phagocytosis failed to promote killing (Harrington-Fowler et al., 1981).
Unlike other intracellular parasites, Listeria sp. is Gram positive and incapable
to mediate interactions with host cells via lipopolysaccharide (Brubaker, 1985).
However, an endotoxin-like material from this organism was recently described that
contained typical chemical markers for lipopolysaccharide (Wexler & Oppenheim,
1979). While this structure probably did not account for typical monocyte-specific
opsonisation with depletion of complement, it may mediate reactions related to
intracellular survival (van Kessel et al., 1981).
In addition, L. monocytogenes is capable to produce a series of toxins, including

haemolysis and lipolysis that are involved in disease expression and manifestation
(Sutherland & Porritt, 1997). The pathogenicity of L. monocytogenes has been linked
with the production of β-hemolysis (Donnelly, 1988). However, the mechanism of
how pathogenic L. monocytogenes are taken up by non-phagocytic cells and its
pathogenicity are poorly understood (Chakraborty et al., 1995).
Virulence genes were identified using molecular genetic techniques include those
involved in entry of L. monocytogenes in epithelial cells (inlA and inlB); escape from
the phagosome (hly and plcA), intra- and extracellular movement (actA), and lysis of
the two-membrane vacuoles (plcB). All of these genes are co-regulated and, except
for the inl genes, are clustered on the same region of the chromosome. In addition,
the enzyme catalase and superoxide dismutase (SOD) may function as secondary
virulence factors. These two enzymes act by degrading toxic oxygen free radicals
produced during the respiratory burst following macrophage phagocytosis (Martin &
Fisher, 2000). Some of the identified virulence determinants in L. monocytogenes is
summarised in Table 8 (Fsihi et al., 2001).
This organism has developed elaborate systems for sensing stresses during the
survival under stress environments, involving a number of changes in the levels of
different proteins and many allied events at the level of gene regulation. There are
relatively little is known of the circuits involved in regulation of stress responsive

Stress and Effects on Virulence of L. monocytogenes / 35
genes by L. monocytogenes. The alternative sigma factor σB has been identified and
sequenced in L. monocytogenes (Wiedmann et al., 1998). This sigma factor appears
to regulate the synthesis of a number of stress responsive proteins. Indeed, BetL, a
glycine betaine transport system linked to salt tolerance of L. monocytogenes has
recently been shown to possess a consensus σB- dependent promoter binding site
(Sleator et al., 1999). An in-frame deletion of a portion of the σB gene eliminates the
ability of this organism to tolerate acid stresses (Gahan & Hill, 1999).
Heat shock linked to the pathogenic virulence is an important aspect in food

system. Heat shock and oxidative stress can induce the expression of listeriolysin, a
protein involved in intracellular virulence of L. monocytogenes (Sokolovic
& Goebel, 1989). This response may correspond to an early phase in the infection
cycle when surviving acidic pH, anoxia, and starvation is more important than the
expression of virulence determinants (Mekalanos, 1992).

Table 8 : Identified virulence determinants in L. monocytogenes

(Fsihi et al., 2001)
Protein Principal characteristics Function
Listeriolysin - 58 kDa, SH-activated, cholestrol- -escape from the phagosome
requiring pore-forming cytolysin -induces phosphorylation of
- pH-dependent hemolytic activity MAP kinases
-induces PtdIns hydrolysis in
HUVEC
Internalin - 84-kDa leucine-rich protein - entry into cultural epithelial
(InlA) - eukaryotic receptor: E-cadherin cells (Caco-2)
- covalently linked to the peptidoglycans - no role identified in vitro
via its LPXTG motif
InlB -67-kDa leucine-rich protein -entry into cultured HepG2,

-surface exposed via its 3 GW modules HeLa, Vero, Hep-2, HUVEC,
and fibroblasts cells
-role in multiplication in
hepatocytes in vivo
-stimulates P13-kinase activity
ActA -67 kDA, polar localization -actin polymerization in

-anchored to the bacterial surface through infected cells
a hydrophobic C-terminal region
PI-PLC -phosphatidylinositol-specific phos- -escape from the primary

pholipase C vacuole in murine
-33 kDa, basic macrophages
-active between pH 5.5 and 7.5 -escape from the double-
membraned vacuole
-amplifies the LLO-related
PtdIns hydrolysis
PC-PLC -phosphatidylcholine-(PC)-preferring -escape from the primary

phospholipase C vacuole in the human
-29 kDa, active between pH 5.0 and 8.0 epithelial Henle 407 cells
-activated on cleavage by the zinc- -escape from the double-
dependent metalloprotease MpI membraned vacuole and cell-
to-cell spread
PrfA -27 kDa DNA-binding protein (CAP/FNR - transcriptional activator of

family) most Listeria virulence genes
ClpC -27 kDa stress protein -intracellular growth in

(Clp/ATPase family) macrophage
InlC -30 kDa leucine-rich protein No defined role, but inlC

mutants show reduced
virulence in mice
Abbreviation used: HUVEC = human umbilical vein endothelial cells, MAP = mitogen activated
protein, PtdIns = phosphoinositides, P13-kinase = phosphoinositide 3-kinase; LRRs = leucine-rich regions

CHAPTER 6
CURRENT METHODS FOR CONTROLLING

L. monocytogenes IN THE FOOD INDUSTRY
The traditional thermal process has been used as a popular and efficient method to
reduce contamination caused by Listeria monocytogenes. Heat resistance of this
organism depends upon many factors including characteristics of different strains
and serovars (Bhaduri et al., 1991; Mackey et al., 1990; Sörqist, 1994). Factors that
affect the susceptibility of L. monocytogenes to thermal treatments include stage in
the growth cycle, temperature during growth, and exposure to other stresses. Cells
in inactive phase and those previously exposed to stresses such as acid, ethanol,
and hydrogen peroxide (Lou & Yousef, 1996) are mostly more resistant to thermal
treatments. Thermotolerance is increased expressively after heat shock (30 min
exposure to 48°C) in cells grown at 4°C (Jørgensen et al., 1999) and tends to
increase in cells grown at higher temperature (Doyle, 1999).
Listeria monocytogenes is not a very heat-resistant organism (Table 9) and

appropriately precise cooking processes using temperature above 70°C will reach
significant reduction in numbers of viable cells. However, heat resistance of L.
monocytogenes is considerably greater than common Salmonella serotypes and
other non-sporing foodborne pathogens, but less resistant than Salmonella senftenberg
775W (Mackey & Bratchell, 1989). Although the differences in heating media,
methods, and test strains, an equation could be derived from a straight line plot
of log D-values versus temperatures based on D-values (decimal reduction time)
obtained from the scientific literature. The equation is: log D = 10.888-0.141519
t, where D is the decimal reduction time in s and t is the temperature (°C), and the z
value is 6.9°C ± 0.136. Many separate estimates of z- values of L. monocytogenes
have been published, ranging from 4.3 to 9.9 (Tomlins & Ordal,i1976).

Table 9: Heat inactivation of L. monocytogenes (Guide to the time and temperature

combination necessary to achieve a 106 reduction (6D) of L. monocytogenes).
Temperature (°C) Time for 6D reduction (min)

63 17
64 12.7
65 9.3
66 6.8
67 5.0
68 3.7
69 2.7
70 2.0
71 1.5
72 1.0
73 0.8
74 0.6
75 0.4
(Source: Food and Drug Administration, et al. (2001)
Although traditional approaches of food preservation are usually used in the

process of food industry, there is substantial concern in new non-thermal technologies
being researched or in the early stages of application. These approaches employ new
technologies alone or synergistically with other novel or traditional means as follows:
6.1 Hurdle technology

This technology is the use of a permutation of different preservation factors
that cannot be elucidated by the microorganisms present in a product. Hurdles
may contain the relations of temperature, water activity, pH, redox- potential and
preservative/antimicrobial agents (Leistner, 1995).
6.2 Natural antimicrobials from microbes, plants or animals

Bacteriocins are proteinaceous, antimicrobial compounds produced by various
kinds of bacteria. Since lactobacilli are known to yield many different bacteriocins
and some are also used in starter cultures for sausage production, addition of
these bacteriocin producers has been effective in reducing L. monocytogenes
population in many fermented meats (De Martinis & Franco, 1998; Holley et al.,
1996; Schillinger et al., 1991).

Current Methods for Controlling L. monocytogenes in the Food Industry / 39
6.3 Food irradiation

Food irradiation can cause mutilation and destroy most foodborne bacteria,
including L. monocytogenes (Fu et al., 1995). Usefulness of a given radiation dose
varies depending on the density, antioxidant levels, moisture, and other constituents
or characteristics of the foods (Doyle, 1999). External factors, such as temperature,
the presence or absence of oxygen, and subsequent storage conditions also affect
the effectiveness of radiation. Some suggested doses of irradiation include: (a) 3 kGy
for elimination of 103 cells of L. monocytogenes per g in air-packed frozen chicken
(Kamat & Nair, 1995); (b) 2.5 kGy to kill about 104 L. monocytogenes per g in
ground beef (Monk et al., 1994); and (c) 2 kGy to destroy 104 L. monocytogenes in
mechanically deboned chicken meat at 2-4°C (Huhtanen et al., 1989).
6.4 High hydrostatic pressure treatment (HHP)

High hydrostatic pressure causes widespread damage to cells with adverse effects
on the membranes, enzymes and other structures and molecules (Mackey et
al., 1994). The effects of high pressure are immediately and consistently transmitted
throughout foods regardless of their geometry of size. Although high pressure
destroys living cells, it does not reduce small molecules like vitamins and flavours
and has negligible effects on the sensory quality of meats (Ananth et al., 1998;
Murano et al., 1999). L. monocytogenes is sensitive to high pressure treatments of
400-500 MPa and some strain variation in sensitivity to pressure is evident at lower
temperatures (25°C) but largely disappears at 50°C (Alpas et al., 1999).
6.5 Pulsed electric field treatment (PEF)

PEF treatment, is a non-thermal process which destroys contaminating bacteria by
short bursts (<1 sec) of high voltage. This technology has been successfully used for
many years to inactivate microorganisms and to extend the shelf life of foodstuffs while
maintaining fresh-like chemical, physical and nutritional properties (MacGregor
et al., 1998). Exposure to PEF destabilizes cell membranes and with sufficient
intensity and duration of treatment, membranes are irreversibly damaged, important
cellular compounds leak out, and cells die (Jeyamkondan et al., 1999; Simpson et
al., 1999). Bacterial spores, Gram-positive cells (including L. monocytogenes), and
cells in stationary phase of growth are more resistant to the effects of PEF (Barsotti
& Cheftel, 1999). For L. monocytogenes suspended in milk, a continuous flow PEF
system resulted in a 3-log10 reduction in bacterial numbers at 25°C and a 4-log10
decrease at 50°C (Reina et al., 1998).

6.6 Ultrasound treatment

Currently, ultrasound is used in food processing for emulsification and accelerating
freezing and cleaning (Earnshaw et al., 1995). Ultrasound kills by disorderly cell
membranes unreliably as a result of the formation and consequent implosion of small
bubbles (cavitation). Because viscous liquids and solids impede the proliferation of
ultrasound waves, this technique is potentially most useful for sterilization of liquids,
such as milk and juices (Pagan et al., 1999).
6.7 Pulsed Ultraviolet light and Continuous Ultraviolet light

Pulsed UV treatment offers the benefits of microbial inactivation with substantially
increased power levels of up to several megawatts (Anderson et al., 2000). Since
UV light cannot penetrate into foods, only microbes on an exposed surface are
susceptible to its deleterious effects. A pulsed Xenon flashlamp was used for
inactivation as this can deliver much more rapid treatment than conventional
continuous UV- exposure. However, the problems of photoreactivation in bacteria
and the possibility of microbes evolving to overcome the biocidal nature of UV
radiation need to be explored (Lani, 2007).
In conclusion, various technologies are available to combat the occurrence

of L. monocytogenes in food industry. However, the efficiency of using these
technologies are depending on intrinsic and extrinsic factors that influence the growth
of this organism. Therefore, this publication covers the overview of this organism and
its implication to food safety. It will help the readers to appreciate the complication
of research dealing with foodborne pathogen.

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INDEX
A G
alternative techniques 5 generation time 21
arc discharge treatment 2 Gram-negative 27, 29
Gram-positive 3, 25, 27, 29, 39
B growth 1-4, 11, 21, 22, 29, 30, 31, 33,
ß-haemolysis 5 37, 39, 40
Bergey's Manual 3, 4
biochemical test 7 H
blood agar 5, 6, 31 heat resistance 19, 22, 24, 25, 26
heat shock 26,35
C high hydrostatic pressure treatment 39
CAMP reaction 7 hurdle technology 38
continuous ultraviolet light 40
conventional 5, 7, 40 I
identification 4, 7
D indicator 21, 30
D-value 20, 21, 25 inhibitory 8, 16, 27, 29
dairy products 12, 19, 22 injured cells 29, 30-32
diagnosis 13 intrinsic 40
diarrhea 12 isolation 8, 9, 11, 30-32
diseases 1, 12, 33
detection 7, 8, 9, 11, 27 K
kinetic models 32
E
Epidemiology 10, 11 L
extrinsic factors 40 lactic acid 16, 18
Listeria monocytogenes 1, 3, 37
F
FDA 8, 9, 10, 12 M
foodborne pathogen 1, 2, 17, 22, 37, 40 modified atmosphere 17
food irradiation 39
food safety 1, 2, 8, 40 N
natural antimicrobials 38
non-selective medium 32
nucleic acid hybridization 7

O T
occurrence 10, 13, 40 taxonomy 3
outbreaks 1, 2, 11-13, 22 temperature stress 26
theory death 19
P thermal inactivation 2, 17, 19-21
pasteurisation 2, 22, 23 thermotolerance 25, 26, 37
pH 1, 4, 16-20, 24, 36
photoreactivation 40 U
pre-enrichment medium 30 unusual resistance 24
pulsed corona treatment 2
pulsed electron beam 2 V
pulsed electric field 2, 39 virulence 2, 13, 15, 16, 33, 34, 35, 36
pulsed ultra-violet (UV) 2
W
R water activity 1, 2, 16, 17, 22, 27, 31, 38
recovery 8, 18, 28, 30, 31, 32
red meat 23, 36 X
repair 27, 28, 30-32 xenon flashlamp 40
reservoirs 10
Z
S z-value 21, 22, 37
salt 17, 18, 28, 35
seafood products 25
sublethal heating 25, 26
sublethal injury 26, 27, 29, 30
survival 2, 15-17, 20, 21, 24, 28, 33
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Listeria monocytogenes: General Overview and Its Signiﬁcance to Food

Book · December 2016

Mohd Nizam Lani Zaiton Hassan

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Microbiological Quality of Ready-to-Eat Foods in Malaysia View project

Environmental Stress and Their Effects on Virulence of Microorganisms View project

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Diterbitkan oleh / Published in Malaysia

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Noor Rohana Mansor, Prof. Madya Dr., 1962-

Reka bentuk: Penerbit UMT

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1 WHY Listeria monocytogenes IS AN IMPORTANT

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Foodborne pathogens continue to cause major public health problems worldwide.

This publication highlights on thermal inactivation of L. monocytogenes.

As intracellular pathogen, L. monocytogenes is able to multiply in the most

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Overall, this publication serves as an introductory resource for those interested

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WHY Listeria monocytogenes IS AN IMPORTANT

Food-borne infections still remain as one of the important causes of preventable

The success of Listeria monocytogenes as a foodborne pathogen is highly

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When environmental parameters are significantly different from the microbial

Exposure to a particular environmental stress may invoke a stress response

Traditional methods of pasteurisation or sterilisation using thermal or chemical

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Listeria monocytogenes is an individual from the genus Listeria and is characterized as

The genus Listeria is classified in Bergey’s Manual of Determinative Bacteriology

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Table 1: Differentiation of Listeria from similar genera

Property Listeria Brochothrix Lactobacillus Kurthia

The genus Listeria was previously classified among “genera of uncertain

2.2 Identification of Listeria Species

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Figure 1: Gram staining of L. monocytogenes cells

Figure 2: Single colony of L. monocytogenes on Blood Agar

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Table 2: Conventional differentiation of Listeria spp.

(Linnan et al., 1988; Jemmi and Stephan, 2006)

Biochemical Confirmation / No 24 hours 6 days 6 days

Immunoassay/ Presumptive Yes 45 minutes to 2 32-50 hours 5-6 days

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2.3 Isolation and Detection of L. monocytogenes

Strategies proposed by some standard associations and other national bodies

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Figure 3: Testing Protocol for L. monocytogenes in Most Food Types

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2.4 Reservoirs of L. monocytogenes

L. monocytogenes is ubiquitous. It has been found in association with a wide

2.5 Occurrence of L. monocytogenes in Food

In Malaysia, Arumugaswamy et al. (1994) examined 234 food samples, consisting

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significant observation in which 22 of the 76 samples of RTE (ready-to-eat) foods

Foods implicated in outbreaks of listeriosis have included products from all of

2.6 Epidemiology of L. monocytogenes

Reported outbreak-associated listeriosis cases represent a small proportion of the

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2.7 Diseases Associated with L. monocytogenes

However, gastroenteritis illness (listerial gastroenteritis) from L. monocytogenes