Detection of Listeria monocytogenes in Pasteurized Milk Sold in Supermarkets in Bogor City and its Relationship with Human Health

DETECTION OF LISTERIA MONOCYTOGENES IN
PASTEURIZED MILK SOLD IN SUPERMARKETS IN BOGOR
CITY AND ITS RELATIONSHIP WITH HUMAN HEALTH

ROBERT KIBUUKA

SCHOOL OF GRADUATE STUDIES
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2009

ISSUES RELATED TO THIS THESIS AND THE
SOURCE OF INFORMATION
With this I declare that this thesis Detection of Listeria monocytogenes in
Pasteurized Milk Sold in Supermarkets in Bogor City and its Relationship with
Human Health is my own work under the direction of an advisory committee. It has
not yet been presented in any form to any Education institution. The sources of
information which is published or not yet published by other researchers have been
mentioned and listed in the references of this thesis.

Bogor, January, 2008

Robert Kibuuka
NIM B251078041

ABSTRAK
ROBERT KIBUUKA. Detection of Listeria monocytogenes in Pasteurized Milk
Sold in Supermarkets in Bogor City and its Relationship with Human Health.
Dibimbing oleh MIRNAWATI SUDARWANTO dan AGATHA WINNY
SANJAYA.
Listeria monocytogenes adalah salah satu bakteri patogen untuk manusia dan hewan
yang ditularkan melalui makanan. Bakteri ini kadang-kadang masih ditemukan dalam
produk yang sudah diolah. Hal tersebut dimungkinkan karena bakteri ini membetunk
biofilm. Kontaminasi Listeria monocytogenes pascapengolahan makanan merupakan
titik kritis untuk kesehatan masyarakat. Penelitian ini dilaksanakan dalam dua cara.
Cara pertama adalah metode kualitatif untuk mendeteksi kehadiran Listeria
monocytogenes dalam susu pasturisasi yang dijual di supermarket. Cara kedua adalah
metode kuantitatif bertujuan untuk melihat gambaran pertumbuhan Listeria
monocytogenes yang ditumbuhkan dalam susu steril dan disimpan pada suhu
rendah(4oC) selama 7 hari. Sejumlah 32 sampel susu pasturisasi yang dibeli dari
beberapa pasar swalayan di Bogor dibiakkan dalam media khusus untuk Listeria sp

(Listeria enrichment broth, Oxford, Tryptose soy agar with yeast extract, Tryptose
soy broth with yeast extract and SIM), dan untuk konfirmasi di lakukan uji CAMP,
katalase, KOH dan uji kimiawi (rhamnose, xylose dan mannitol) serta pewarnaan
GRAM. Hasil yang diperoleh dengan metode kualitatif menunjukkan hasil negatif.
Hasil penelitian dari metode kuantitatif belum menunjukkan pertumbuhan Listeria
monocytogenes pada hari pertama, kedua, ketiga dan keempat. Pada hari kelima
pertambahan jumlah bakteri ini meningkat dengan tajam. Hal ini mengindikasikan
bahayanya produk olahan susu yang terkontaminasi oleh Listeria monocytogenes
walaupun disimpan dalam suhu rendah.
Kata kunci: Listeria monocytogenes, susu pasturisasi, jumlah coloni, kesehatan
manusia.

SUMMARY
ROBERT KIBUUKA. Detection of Listeria monocytogenes in Pasteurized Milk Sold
in Bogor City and its Relationship with Human Health. Under direction of
MIRNAWATI SUDARWANTO and AGATHA WINNY SANJAYA.
Listeria monocytogenes is a foodborne bacterium recognized as pathogenic
for both humans and animals. Because of its versatility, Listeria is able to persist in
the food industry environment for several years, probably in a biofilm state. Post –
processing contamination of food with L.monocytogenes is a critical problem of

public health. Several outbreaks of listeriosis were linked with the consumption of
minimally processed and ready to eat (RTE) foods. Because of this, many countries
have established a zero tolerance policy, under which RTE foods contaminated with
L.monocytogenes at a detectable level are deemed adulterated.
It is a Gram-positive bacterium, non-spore forming bacilli. Listeria is aerobic
or facultative anaerobic, catalase positive and oxidase negative and tolerates low
water activity. It is also capable of growing between 4oC to 37oC and has a unique
tolerance to low water activity. Temperature of 4oC is generally regarded as safe for
storage of foods. Pasteurization is a world-wide heating process used to reduce
bacterial populations present in milk. The two methods include high temperature
short time (HTST) at 71.7oC for 15 seconds and low temperature long time (LTLT) at
61.7oC for 30 minutes.
The research was done in two parts. The first part was to qualitatively identify
the presence of Listeria monocytogenes in pasteurized milk sold in different
Supermarkets in Bogor City. The method was adopted from the Bacteriological
Analytical Manual / Food and Drug Administration (FDA 2003). All the samples
tested negative to Listeria monocytogenes. The second part of the research was to
evaluate the growth of L. monocytogenes in sterile milk stored in the refrigeric
incubator set at 4oC and monitored for 7 days. The original L .monocytogenes culture
at a concentration of 1x109cfu/ml was diluted with Buffered Phosphate Water (0.1%)

to achieve a cell concentration of approximately1.0 x 102 cfu/ml. Eight clean and
sterile erlenmeyers were aseptically filled, and then a 0.1ml of it was aseptically
pippeted into each of the seven erlenmeyer with codes E1 to E7. E8 was not
inoculated with L. monocytogenes because it was the control sample. All the
erlenmeyers were stored in the refrigerator set at 4oC. Growth was monitored on
nutrient agar plates incubated at 37oC for 24 – 48 hours. Colonies were then counted
using the quebec colony counter. Listeria monocytogenes maintained its population
relatively well in sterile milk stored at 4°C. Growth was observed on the first, second,
third, fourth and fifth day. On the sixth day and the seventh day, the numbers of
colony forming units observed were almost similar. A population of 10cells is enough
to cause serious listeriosis in humans.
Kata kunci: Listeria monocytogenes, susu pasturisasi, jumlah coloni, kesehatan
manusia.

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DETECTION OF LISTERIA MONOCYTOGENES IN
PASTEURIZED MILK SOLD IN SUPERMARKETS IN BOGOR
CITY AND ITS RELATIONSHIP WITH HUMAN HEALTH

ROBERT KIBUUKA

A thesis
as part of the requirements for achieving the degree of
Master of Science in the
Veterinary Public Health Study Program in the Department of Animal Diseases and
Veterinary Public Health

SCHOOL OF GRADUATE STUDIES
BOGOR AGRICULTURAL UNIVERSITY
BOGOR

2009

Thesis External Examiner: Dr. Drh. Denny W. Lukman, MSi

Tittle of Thesis

:

Name

:

Detection of Listeria monocytogenes in Pasteurized
Milk Sold in Supermarkets in Bogor City and its
Relationship with Human Health
.
Robert Kibuuka

Registration number

:

B251078041

Study Programme

:

Veterinary Public Health

approved by supervisors

Prof.Dr.Drh. Hj. Mirnawati Sudarwanto
Head

Dr. Drh. A.Winny Sanjaya, MS
Member

acknowledged by
Head of Study Program

Dean of Graduate School

Dr. Drh. Denny W. Lukman, MSi

Prof. Dr. Ir. Khairil A. Notodiputro, MS

Date of Examination: 13/01/ 2009

Date of Graduation…………2009

ACKNOWLEDGEMENTS
Several people in one way or another contributed to the success of this work. I
would like to convey my sincere and special thanks to my main supervisor Prof. Dr.
Drh Hj. Mirnawati Sudarwanto for her guidance, encouragement, advice and
constructive criticisms during the preparation, survey sampling, laboratory analysis
and finalizing this thesis. I would like also to thank her for allowing me carry out my
research using her media and reagents and later I paid back in installments, otherwise
this work would not have been completed in time. May the Almighty God reward her
more.

I wish also to thank my second supervisor Dr. Drh Agatha Winny Sanjaya
MS, for her valuable time and comments on this work. Her contribution during
laboratory analysis cannot be underestimated. I am very grateful to the Indonesian
government that gave me the opportunity to do a Master of Science in Veterinary
Public Health from this prestigious University of Bogor Agricultural University under
the Developing Countries Partnership Program (KNB).
It’s also with sincere gratitude that I acknowledge the contributions of Dr Drh
Denny Widaya Lukman MSi, in his capacity as the head of the study program for his
guidance and encouragement throughout my study period, more especially at the time
when I was still learning the Indonesian Language. Special thanks also go to the
following people:
1. Drh Trioso Purnawarman, MSi. for accepting me carryout my analysis in the
Laboratory of Veterinary Public Health , Faculty of Veterinary public Health
Bogor Agricultural University , Bogor , Indonesia.
2. Drh. R.Roso Soejoedono, MPH, DEA for his advice and always making me
feel comfortable in the University.
3. The technicians in the laboratory, Mr. Tedi and Mr. Yuhendra who were
always there to help and advise me in all laboratory aspects. I will never
forget them.

Special thanks go to my Parents, Mr. and Mrs. George William Mumira, my brothers
and sister for their continued prayers. Irene Nassazi, Raymond Ddamba and Carolyn
Nansubuga that you people mean so much to me and I will always be there for you
just the way you have always been there for me. Lastly, to all my classmates Elfa
Zuraida and Umi Siti Saleh and all my other friends not forgetting Drh Andrijanto
MSi that I have learnt a lot from you people, thank you so much.
Bogor, January 2009

Robert Kibuuka

CURRICULUM - VITAE
The writer was born on 23rd January 1972 from Mr. and Mrs. George William
Mumira of Wamala Village in Entebbe, Wakiso District, Uganda. The writer is the
eldest in the family of nine children. The writer went to Kabulamuliro Primary
School in Entebbe, Wakiso District. In 1986 he joined Gombe Secondary School in
Mpigi District for his ordinary level studies. In 1990 he joined Caltec Academy
Makerere for his advanced level studies and in 1994 he joined Makerere University
Faculty of Veterinary Medicine. In the year 2000, he graduated with a bachelor’s
degree in Veterinary Medicine. Since then he has been working as a veterinary officer
in Rakai district, under the Ministry of Agriculture, Animal Industry and Fisheries of

the Republic of Uganda. In 2006, the writer was offered the opportunity by the
Indonesian government under the developing countries partnership scholarship for
two years to do a Master of Science in Veterinary Public Health in Bogor
Agricultural University, Indonesia.

LIST OF CONTENTS
Page
LIST OF TABLES

....…........…………............………………………...…

ii

LIST OF FIGURES

........... ………………...................…………………..

iii

LIST OF APPENDICES

...........………………………............……….......

iv

I. INTODUCTION
Background .............………………..……..........................…............
Problem Statement ..............…….………..…….....................….........
Objectives of Research ...........................…………………….............

1
3
3

II. LITERATURE REVIEW
History of Listeria monocytogenes .....................................................
Listeria monocytogenes and the Disease ..............................................
Growth of Listeria monocytogenes in Cold Storage ...........................
Sources of Listeria monocytogenes in Retail Shops ............................
Listeria monocytogenes and its Characteristics ..................................
Occurence and Ecology of Listeria monocytogenes ...........................
The Disease in Man ..............................................................................
Control of Listeria monocytogenes in Foods..........................................
Chromogenic Medium for Isolation of Listeria monocytogenes ..........

4
4
5
6
8
11
13
14
15

III. MATERIALS AND METHOD
Time and Place .....................................................................................
Materials ...............................................................................................
Media and Chemical................................................................................
Equipments..............................................................................................
Sample Handling after Collection...........................................................
Qualitative Analysis of Listeria monocytogenes.....................................
Innoculation of Listeria monocytogenes in Sterile Milk.........................
Data Management and Analysis..............................................................

17
17
17
17
18
18
19
22

IV. RESULTS AND DISCUSSION
Qualitative Findings................................................................................
Quantitative Findings..............................................................................
Effect on Human Health..........................................................................

23
25
27

V.

CONCLUSION AND RECOMMENDATION
Conclusion...............................................................................................
Recommendations...................................................................................

REFERENCES.................................................................................................
APPENDIX......................................................................................................

29
29
30
34

i

LIST OF TABLES
1
2
3
4
5

Biochemical Differentiation of Listeria species................................
Counts of Listeria monocytogenes in Milk at 4oC for 7 days...........
Listeria moncytogenes Inactivation Temperature.............................
Results of Detecting the Presence of Listeria monocytogenes in Milk......
Total Colony Counts for Listeria monocytogenes in Sterile Milk................

Page
9
10
10
23
25

ii

LIST OF FIGURES

1
2
3
4
5
6
7
8
9
10
11

Scanning Electron Micrograph of Listeria monocytogenes..............
Consumers Carry the bacteria on Clothes while Shopping...
Schematic Representation of a Milk Suply Chain............................
Listeria monocytogenes Gram Staining............................................
Growth and Valiability of Listeria monocytogenes in Certain
Foods at Freezing ...........................................................................
Ways in which Listeria monocytogenes is Disseminated in the
Environment, Animals and Humans.................................................
Listeria monocytogenes Pathways into the Food Processing Plant...
Morphology of Listeria species Growth on ALOA Agar Plates.......
Flow Diagram for the Qualitative Isolation of Listeria
monocytogenes Foods.......................................................................
Milk Samples for Quantitative Analysis of Listeria monocytogenes
kept in Refrigerated Incubator set at 4oC..........................................
Regression Curve for Growth of Listeria monocytogenes in Sterile
Milk Stored in Refrigerator at 4oC for 7 Days..................................

Page
5
6
7
8
10
11
13
15
18
22
26

iii

LIST OF APPENDICES

1
2
3
4

Sampling Plan...................................................................................
List of Abbreviations.........................................................................
Flow Diagram for Qualitative Analysis of Listeria monocytogenes
growth in an Innoculated Milk sample in Nutrient Agar..................
Regression Analysis for Listeria monocytogenes growth in Sterile
Milk Stored in Refregerator at 4oC for 7 Days..................................

Page
35
35
36
37

iv

INTRODUCTION
Background
Milk is a food material which contains proteins, fats, lactose, minerals,
vitamins and enzymes. It is produced by the mammary glands in the udder under
the influence of certain hormones. It is a food material that is destined to satisfy
nutritional requirements and is consumed in a modified, prepared or treated form
through drinking, eating or intake in any other form. Milk always contains a few
microorganisms. Contamination comes from the cow, equipments, unhygienic
human handling, unclean storage rooms, and milking tools (Volk and Wheeler
1990). For human consumption it must fulfill the requirements for safe foods
since it is already proved that milk is one of the vehicles through which
pathogenic microorganism can be transferred into the human body (WHO 2002).
Some of the pathogenic microorganisms that contaminate milk and make it unsafe
for consumption are Brucella sp., Bacillus cereus, E. coli O157:H7,
Campylobacter sp. Staphylococcus aureus, Salmonella sp. and L. monocytogenes
that causes listeriosis are important food borne diseases that have emerged over
the last decades. Milk has specific characteristics that support the growth of
microorganisms and these include; higher water activity (a w) of 0.993,
temperatures of 25oC, a neutral pH of 6.6-6.9 and presence of large quantities of
proteins (Anonymous 2001).
According to WHO (2002), it was reported that in 2001 alone, 2.1 million
people died from diarrhoea diseases and a great proportion of these cases was
attributed to contamination of foods and drinking water. In industrialized
countries, the percentage of people suffering from food borne diseases each year
has been reported to be up to 30% and in the USA alone, around 76 million cases
of food borne diseases resulting in 325,000 hospitalizations and 5000 deaths are
estimated to occur each year. While less documented, developing countries bear
the brunt of the problem due to the presence of a wide range of food borne
diseases including those caused by parasites. The high prevalence of diarrhoea
diseases in many developing countries suggests major underlying food safety
problems.

2

Listeria monocytogenes is one of those bacteria that have been involved in
these outbreaks. It causes a disease known as listeriosis as a result of consumption
of contaminated foods. The disease mainly affects pregnant women, new borne
babies and adults whose immune system is very low such as in cancer and
HIV/AIDS victims. It is a fatal disease with mortality rate of 25% (compared to
mortality rate of 1% for salmonella), and hospitalization rate is 92 % (Anonymous
2001). The organism causes two forms of the disease; invasive and non-invasive.
The invasive disease normally occurs in people with weakened immune system
while the non-invasive disease can occur in anyone if a high number of Listeria
monocytogenes cells are consumed. The invasive form has an incubation period of
1-90 days following consumption of 102 to 103 cells and characterized by flu-like
symptoms, meningitis, septicemia, diarrhea, vomiting and spontaneous abortion.
The incubation period for the non-invasive form is 11 hours to 7 days following
consumption of 105-1011 cells, characterized by diarrhea, fever, muscle pain,
headache, and less frequent abdominal cramps and vomiting. It has also been
termed as febrile gastroenteritis. Anonymous (2001) reported that in one outbreak
neurological problems (cranial nerve palsies) developed in 30% of the survivors
of meningitis. Pre- term infants may suffer from excess fluid in the brain.
The sources of Listeria monocytogenes include humans, animals, food and
the environment. In humans, Listeria monocytogenes is carried asymptomatically
in the feces of 2-6% of the population. It is shed in high numbers (>10 4/g) in feces
of infected people (Anonymous 2001).
Like in humans, animals too are affected and veterinarians are considered to
be a high risk group. Listeria present in the animal feces can contaminate milk and
other animal products and improperly made silage can be a source of domestic
animal infection. The organism is also considered to be potentially present in all
raw foods and ingredients. Risk posed is likely to be greatest in ready-to-eat foods
with long shelf lives. However, pasteurization is an important process used to
reduce bacterial populations present in milk hence safe for consumption. The two
methods include high temperature short time (HTST) at 71.7oC for 15 seconds
and low temperature long time (LTLT) at 61.7oC for 30 minutes.

3

Problem Statement
Food borne illness is recognized as a significant public health problem
throughout the world, though data is incomplete that would permit accurate
quantification of morbidity and mortality associated with food borne hazards
especially in developing countries. Much of the burden of illness were as results
of basic sanitation failures that occur in food production, processing, retailing, and
handling at home. For processed foods, Uganda depends heavily on imports and
must be protected from products that are sub-standard in quality or beyond their
expiration date. Consequently, expansion for production and export of valueadded products in developing countries are strategic keys for future economic
development. This will require the implementation of reliable in-plant HACCPbased quality and safety control systems. There is failure of many African
produced food products to meet the international food-safety and quality standards
thereby hampering the continent’s efforts to increase agriculture trade both intraregionally and internationally, locking many farmers out of a chance to improve
their economic well-being.
Objectives of the Research
a. The objective of this research was to detect the presence of L.
monocytogenes in different brands of pasteurized milk sold in
supermarkets in Bogor City.
b. To determine the number of colony forming units per ml of L.
monocytogenes in the sterile milk sample after 7 consecutive days in the
refrigerator at 4oC and relate it to the health of the consumer.
The hypotheses of the research were that there is Listeria monocytogenes in
pasteurized milk sold in supermarkets in Bogor City and the second was that there
is no Listeria monocytogenes in pasteurized milk sold in supermarkets in Bogor
City.

4

LITERATURE REVIEW

History of Listeria monocytogenes
The history of L. monocytogenes begun in 1924, when Murray, Webb and
Swann (1926) isolated a 1-2µm long and 0.5µ wide round-ended Gram- Positive
rod in dead laboratory rabbits and guinea-pigs in Cambridge and named it
Bacterium monocytogenes. In 1927, Pirie described an unusual death of gerbils in
South Africa and named it Listerella hepatolytica in honor of Lord Joseph Lister.
The two strains isolated by Murray et al. (1926) and Pirie (1927) showed
great similarity, the bacterium was renamed Listerella monocytogenes. However
the generic name Listerella had previously been used for protozoan and Pirie
(1940) proposed changing the name to L. monocytogenes. This name was
accepted although it already existed in Botanical taxonomy, an orchid named
Listeria and in zoology a diptera called Listeria (Seeliger 1961). Genera of
Listeria and Brochothrix are members of the family Listeriaceae, the order
Bacillales, the class Bacilli and phylum Firmucutes.

Listeria monocytogenes and the Diseases
In order for food to reach the consumer, it goes through a long way until it
reaches the retail shops. Even with modern food processing equipments,
production of contaminated foods is still possible and this usually takes place at
the time of production, processing, distribution and storage. From 1983 up to
2003, more than 300,000 cases were reported by Centers for Disease Control and
Prevention (FDA 2006) to have taken place as a result of consumption of
contaminated foods. In the United States of America, data available indicates that
76 million who were affected, 5000 of them died due to consumption of
contaminated foods and L. monocytogenes was implicated in the outbreak.
Listeria monocytogenes is a food borne bacterium recognized as pathogenic
for both humans and animals. Because of its versatility, Listeria is able to persist
in the food industry environment for several years probably in a biofilm state
(Moretro et al. 2007). Post-processing contamination of food with L.
monocytogenes is a critical problem of public health. Several outbreaks of

5

listeriosis were linked with the consumption of minimally processed and ready to
eat (RTE) foods (FDA 2003).

Figure1 Scanning electron micrograph of Listeria monocytogenes (FDA
2006)
Listeria monocytogenes compared to other food borne diseases cause fewer
outbreaks but usually causes 20% deaths (FDA 2003). Listeria monocytogenes is
found everywhere in the air, plants and animals. Fruits and vegetables that are not
properly washed can easily contaminate those other foods to which they are
added, for example salads consumed together with meat or sausages.

Growth of Listeria monocytogenes in Cold Storage during Retail
One of the characteristics of Listeria monocytogenes is its ability to grow
in cold temperatures, including foods stored in the cold for longer periods of time.
Retailing of foods requires that they use standard temperatures (41 oF) for storage
of ready to eat foods or below the above temperature or heating the food
suspected have been contaminated by Listeria monocytogenes (FDA 2007). It is
also important to take care of the environmental conditions surrounding the place
where the food is stored in retail places in order to minimize the factors that can
lead to occurrence or growth of Listeria monocytogenes for example the food
washing room, the person assigned the duty of washing the food, protective
clothing and also monitoring the sanitation.

6

Source of Listeria monocytogenes in Retail Shops
Generally, the source of Listeria monocytogenes in retail shops includes
food products, environment, equipments, owner, the consumer or the user of the
product. Several food products sold in retail shops must have standard operating
procedures (SOPs) to protect foods from getting contaminated by bacteria or other
pathogens before they are sold to the consumer. Raw materials from meat,
chicken, sea- foods, fruits or vegetables carry some pathogenic bacteria including
Listeria monocytogenes. Dust or tools that are contaminated can transmit Listeria
monocytogenes. Retail workers and consumers can also contaminate the foods by
carrying the bacteria. In this situation, the bacteria are carried on clothes, shoes or
hands that are contaminated at the time of selling and buying (Figure 2).

Figure 2 Consumers can carry the bacteria on their clothes or shoes at the
………….. time of shopping (USDA 2006).

Available reports highlight the importance of cross-contamination of
processed foods from the environmental sources. Listeria is widely disseminated
in the rural environment and consequently milk can be contaminated at any stage
from farm to table as can be seen in the schematic milk supply chain (Figure3).

7

Raw milk receiving

Filter

Raw milk storage

Clarifier or Separator

Homogenized

Pasteurization

Filler

Raw cream
storage
Vitamin
addition
Packaging
material

Cold storage

Distribution

Consumer

Figure 3 Schematic representation of a milk supply chain (Elmer et al. 2001).
It is still unknown if its persistence in the processing environment is as a
result of adaptation of certain subtypes of the organism, or poor cleaning and
disinfection, or the ability of the microorganism to develop tolerance to some of
the used products. In general, adherent bacteria appear less sensitive to cleaning
and disinfection than bacteria in suspension (Moretro et al. 2007) and indeed
adherent listeria cells are more resistant to biocides than suspended listeria cells.
The susceptibility of listeria cells to biocides may also be reduced by the presence
of soil on the surface (Kornacki et al. 2000).

8

Listeria monocytogenes and its Characteristics
It is a Gram-positive bacterium, non-spore forming bacilli. Listeria is
aerobic or facultative anaerobic, catalase positive and oxidase negative and
tolerates low water activity. It is also capable of growing between 4oC to 37oC and
has a unique tolerance to low water activity (Tienungoon et al. 2000).
Temperature of 4oC is generally regarded as a safe temperature for storage of
foods and 37oC is a body temperature that can favour growth of L. monocytogenes
when consumed in foods. Other types include Listeria innocua, Listeria ivanovii,
Listeria seeligeri, Listeria welshmeri, and Listeria grayi.

Figure 4 Listeria monocytogenes Gram Stain (Anonymous 2003).

Species difference is based on the above mentioned activities as can be
seen in Table 1

9

Table 1 Biochemical differentiation of Listeria species (FDA 2003)

According to Doyle et al. (2001), L. monocytogenes grows in pasteurized
milk, with the numbers increasing 10-fold in 7 days at 4oC. Grows more rapidly
in pasteurized milk than in raw milk when incubated at 7oC. Therefore, fluid milk
that becomes contaminated after pasteurization and stored under refrigeration
temperature may attain very high populations of L. monocytogenes after one
week. Therefore temperature abuse may further enhance the multiplication of
bacterial cells. In relation to the above condition, Hayes (1996) noted that the
organism exhibits a surprising resistance to the heat and it has been suggested that
it survives the minimum pasteurization heat treatment of 72 oC for 15 seconds
required by many countries for raw milk. It is the most heat resistant bacteria
among those that do not form spores (Lou and Yousef 1999). This point is
illustrated in table 2, 3 and Figure 5.

10

Table 2 Counts of Listeria monocytogenes in milk (initial count 108 per ml)
stored at 4oC for 5 days after pasteurization (Hayes 1996)
Immediately after
pasteurization and storage at
4oC

Days
0
I
II
III
IV
V

cfu/ml
0
0
40
150
800
2500

Table 3 Listeria monocytgenes inactivation temperatures (FDA 2003)

Figure 5 Growth and viability of Listeria monocytogenes in certain foods at
..freezing temperatures (-20oC) and refrigeration temperatures (4oC)
. .over a 12 week period (Anonymous 2008).

11

Occurrence and Ecology of Listeria monocytogenes
Listeria monocytogenes occurrence in foods, food processing plants and
environment (Figure 6). Environmental food regulatory agencies in many
countries have accepted that it is impossible to produce Listeria monocytogenes
free ready to eat foods and have established tolerance levels for L.
monocytogenes. These countries include among them Canada and France. While
in the United States of America (USA) and Britain, although they acknowledge
the ubiquitous distribution of Listeria monocytogenes in food supply and the
difficulties in producing listeria free ready to eat foods, have decided not to adopt
tolerance levels for Listeria monocytogenes in these foods. Both countries argue
that any acceptable levels for Listeria would require knowledge of the number of
organisms unlikely to cause infection and therefore have a zero tolerance to L.
monocytogenes in these foods (Shank et al. 1996). A schematic representation of
the ways Listeria monocytogenes circulates between man, animals and the
environment is as indicated in Figure 6.

ANIMAL SECRETION AND
EXCRETION
Feces, uterine and vaginal secretions and
placenta

ANIMAL
Disease/Intestinal
Colonization

Aerial
Contamination

Sewage

ENVIRONMENT
SOIL--------WATER
Vegetable
(Cabbage, plants, silage)

Aerial
Contamination

Sludge, water,
river

MAN
Disease/
Intestinal
Colonization

ANIMAL PRODUCTS
Meat, Milk, Carcasses, Dairy
products

Figure 6 Ways in which Listeria monocytogenes is disseminated in the
environment, animals, foods, and humans (Doyle et al. 2001).

12

The genus Listeria can be isolated from plants, sewage and water and from
feces of man and animals that look healthy. Epidemiological data from several
countries indicate that listeriosis in humans is related to three serotypes of L.
monocytogenes 1/2a, 1/2b, and 4b (Jemmi & Stephan 2006). This condition
indicates that there is a big adaptation of certain sub-types of L. monocytogenes in
several foods, environment, and humans. It is now also considered to be an
indicator bacterium in industrial food processing chain.
Entry of L. monocytogenes into food processing plants occurs through soil
on workers shoes and clothing and on transport equipment, animals which excrete
the bacterium or have contaminated hides or surfaces, raw plant tissue, raw food
of animal origin and possibly human healthy carriers (Fenlon et al. 1999). Growth
of L. monocytogenes is favored by high humidity and presence of nutrients. Quite
often it is detected in moist areas such as floor drains, condensed and stagnant
water, floors, residues and processing equipment. It can attach to various kinds of
surfaces (stainless steel, glass, and rubber) and biofilms have been described in
meat and dairy processing environments (Shank et al. 1996). It can also survive
on fingers after hand washing and aerosols. The presence of L. monocytogenes in
the food processing chain is evidenced by the wide spread distribution of listeriae
in processed products. Sources of L. monocytogenes in dairy processing plants
include the environment (floors and floor drains, especially in areas around the
coolers or places subject to outside contamination) and raw milk. Efforts to ensure
that milk is safe from L. monocytogenes contamination should focus on
identifying and eliminating sources of post pasteurization contamination
(Tompkin et al. 1999). This is the most likely route of contamination of processed
foods by L. monocytogenes. It is a very difficult bacterium to eliminate from the
food processing plant because it has the propensity to adhere to food contact
surfaces and to form biofilms, which makes implementing effective sanitation
procedures difficult. The refrigerated, moist environments in food processing
plants provide a good growth environment for L. monocytogenes (Petran &
Zottola 1989). Because L. monocytogenes is a frequent contaminant of raw
materials used in food processing plants, there are ample opportunities for re-

13

introduction of Listeria into food processing facilities. Figure 7 indicates L.
monocytogenes pathways into the food processing plant.

Figure 7 Listeria monocytogenes pathways into the food processing plant.
Pasteurization is a world-wide heating process used to reduce bacterial
populations present in milk to between 4-50%. The two methods include high
temperature short time (HTST) at 71.7oC for 15 seconds and low temperature long
time (LTLT) at 61.7oC for 30 minutes. The aim of food processing and storage is
to limit the opportunity for microorganisms to grow to the point where they are
able to cause milk spoilage or to the point where they can cause food to be unsafe
for human consumption (Tienungoon et al. 2000).
The Disease in Man
The most affected age group is newborns followed by those of the over
age 50. The disease is very rare between 1month and 18 years of age. According
to data, listerial abortion usually occurs in the second half of pregnancy and it is
more frequent in the third trimester. Symptoms that precede miscarriage or birth
by a few days or weeks may include chills, increased body temperature,
cephalalgia, slight dizziness and sometimes gastrointestinal symptoms (Elmer et
al. 2001). These septicemic episodes may or may not recur before birth of a
stillborn fetus or a seriously ill full-term baby. After delivery the mother shows no

14

disease symptoms, but L. monocytogenes can be isolated from the vagina, cervix
and urine for periods varying from a few days to several weeks. If a child is born
alive but was infected utero, it may show symptoms immediately after birth or
with in a few days. The symptomatology is that of sepsis or less frequently a
disseminated granulomatosis. There may also be symptoms of a respiratory tract
disorder. Case fatality is high. The main lesion is a focal hepatic necrosis in form
of small, grayish-white nodules. Some children apparently born healthy fall ill
with meningitis shortly thereafter. Meningitis or meningoencephalitis is the most
common clinical form in adults, especially in those over 50 years. Listerial
septicemia usually occurs among weakened adults, especially patients undergoing
long- term treatment with corticosteroids. The recommended treatment for
maternal – fetal listeriosis is ampicillin. Various antibiotics, such as ampicillin
alone or in combination with aminoglycosides, tetracycline (not used for those
under 8 years of age), and chloramphenicol, may be used for other forms of the
disease (Slutsker et al.1990).

Control of Listeria monocytogenes in Food
Control measures should surmount the problems associated with the
ability of L. monocytogenes to withstand relatively high temperature , its ability to
grow down to just over 0oC and its ability to so effectively repair from the effects
of metabolic injury at low temperatures.It is therefore essential to ensure that milk
is adequately pasteurized and there is no post – pasteurization contamination,
ensure foods are heated to a minimum internal temperature of 72 oC and ensure
that only L.monocytogenes - free milk is used in the manufacture of foods. In
addition to the above measures, its important to ensure that the shelf life of all
products, heated or unheated, where growth is feasible should be limited to a
maximum of five days. Much as L. monocytogenes is capable of growing at low
temperatures, the maximum temperature to keep all ready to eat foods must be
maintained at 4oC.

15

Chromogenic Medium for Isolation of Listeria monocytogenes
A microbiological media that has the ability to differentiate between the
different types of Listeria is so important in the isolation and identification, for
example lactose in MacConkey Agar and hydrogen sulfide (Anonymous 2003).
These tests are less specific and give an impression of false positives or negative.
At the moment, chromogenic substrate has the best ability to differentiate
the pathogenic bacteria specifically, including E. coli, coliforms, Listeria spp. and
Salmonella. More so, it is very effective in isolation of Listeria monocytogenes
for example ALOA agar (AFNOR Validated Method), very specific and sensitive
in a longer time, but besides that, it is far better compared to other media that are
always used for isolation of Listeria monocytogenes. It also minimizes the growth
of other contaminating microorganisms, using lithium chloride as anti-micro
bacteria (Anonymous 2001) and it contains a mixture of anti- fungi. Its other
components include a chromogenic substrate namely X- glucosidase. Listeria spp.
that grows in this media produces blue-green colonies following incubation at
37oC for 24 hours. Meanwhile, non-Listeria grows with white colonies (Figure
8).

Typical colonies of
L. monocytogenes

Typical colonies of
L. ivanovii

Typical colonies of
L. innocua

Figure 8 Typical morphology of Listeria Spp. growth on ALOA agar plates
(Anonymous 2001).
According to FDA/BAM (2003), genus Listeria has six species namely L.
monocytogenes, L. innocua, L. seeligeri, L. welshimeri, L. ivanovii and L. grayi.
From all these, Listeria

monocytogenes is the most pathogenic to man if it

contaminated foods or drinks that are consumed by individuals. It is a Gram-

16

positive bacterium, non-spore forming bacilli. Listeria monocytogenes is aerobic
or facultative anaerobic, catalase positive and oxidase negative. It is capable of
growing between 4oC to 37oC and has a unique tolerance to low water activity
(Tienungoon et al. 2000). Temperature of 4 oC is generally regarded as a safe
temperature for storage of foods but it is violated by the psychrotrophic Listeria
monocytogenes bacteria.
Doyle et al. (2001) reported that L. monocytogenes grows in pasteurized
milk, with the numbers increasing 10-fold in 7 days at 4oC, and it grows more
rapidly in pasteurized milk than in raw milk when incubated at 7 oC. Therefore,
fluid milk that becomes contaminated after pasteurization and stored under
refrigeration temperature may attain very high populations of Listeria
monocytogenes after one week. Therefore temperature abuse may further enhance
the multiplication of bacterial cells. In relation to the above condition, Hayes
(1996) noted that the organism exhibits a surprising resistance to the heat and it
has been suggested that it survives the minimum pasteurization heat treatment of
72oC for 15 seconds required by many countries for raw milk. It is the most heat
resistant bacteria among those that do not form spores (Lou & Yousef 1999).

17

MATERIALS AND METHODS
Time and Place
This research was carried out from June up to August 2008 in Laboratory of
Veterinary Public Health, Faculty of Veterinary Medicine, Bogor Agricultural
University (IPB) in Bogor city west Java Indonesia.
Materials
Samples collected were those of pasteurized High Temperature Short Time
(HTST) liquid milk locally processed and packed in Indonesia. In Bogor City,
there are several supermarkets that sell different brands of pasteurized milk from
different factories in Indonesia. A total of 32 samples were collected from four
different big supermarkets in Bogor City. They were in four different brands,
locally manufactured and each brand was picked eight times.
Media and Chemicals
These included Listeria enrichment broth (LEB), Modified Oxford
medium, Tryptose soy agar-yeast extract (TSA-YE), Tryptose soy broth-yeast
extract (TSB-YE), CAMP test cultures (Staphylococcus aureus ATCC 25723,
Sheep blood agar), Gram stain kit, Hydrogen peroxide (H2O2) 3%, Mannitol,
Maltose, Rhamnose , Xylose and Nutrient agar.
Equipments
These included Cover slips, Erlenmeyer flasks (500 ml), Immersion oil
(for microscope), Incubators (30oC + 2oC and 37oC +2oC), Refrigeric incubator
(4oC+2oC) Inoculating loops (Ose), Microscope, Vortex mixer, Electric Lamp,
Petri dishes, Pipettes (1.5 and 10 ml), Scissors, Autoclave, Quebec colony
counter, Grease pencil or Marker and Test-tubes.
Sample Handling after Collection
Samples were placed in insulated cooler containing ice packs, which were
placed on the sides, in the middle and on top of the product. More ice was always
added to ensure that the temperature is within the range of 4 °C to 10 °C. Proper
sterilization of all the equipments, disinfection of the outside of the packs before
opening and hand washing and disinfection using alcohol (70%) before opening
were done to ensure that there was no external contamination.

18

Qualitative Analysis of Listeria monocytogenes
The method used for detection of Listeria monocytogenes was adopted
from the Bacteriological Analytical Manual / Food and Drug Administration
(FDA 2007). The general schematic sample testing is as shown in Figure 9.
Milk Sample
(10 ml)

LEB (100ml)
Mix

Incubation 300C, 24,48hrs,
until 7 days
and Streak

Oxford (24 - 48 hours), 37 oC
After growth, pick 5
suspected black colonies
(black

TSAYE
(incubation for 24 hours, 37 oC

KOH test

Catalase test

CAMP Test

Gram –Staining

TSB YE
Sugar
(Mannitol, Xylose, Rhamnose)

Figure 9 Qualitative Isolation of Listeria monocytogenes

SIM

19

Isolation of Listeria monocytogenes from dairy products was based on the
FDA protocol. It begins with enrichment of the sample in Listeria Enrichment
Broth (Oxoid M0897) a buffered medium supplemented with SR141 (contains
acriflavin, nalidixic acid, and cyclohexidine as selective agents). After 24 and 48 h
of incubation at 30oC, the enrichment culture was streaked to a Listeria selective
plating media called Oxford medium (OXA) which contains polymyxin B
acrilflavin, and ceftazidime. After 24 – 48h and until seven days of incubation at
37oC, suspect colonies OXA are black halo resulting from esculin hydrolysis after
which the five suspect colonies were streaked into Tryptose Soy Agar with yeast
extract (TSA Oxoid CM) 131 and incubated at 37oC for 24h. Presumptive Listeria
isolates are speciated based on a standard series of biochemical tests. Typical
L.monocytogenes isolates are

rhamnose positive, xylose negative, and CAMP

test positive with β-hemolysis enhanced in the vicinity of Staphylococcus aureus.
The suspected colonies were also subjected to potassium hydroxide, motility test
using SIM medium (Oxoid CM0435) for detecting the umbrella-like growth,
Hydrogen peroxide, and mannitol. Colonies from Tryptose Soy Agar with Yeast
extract were again re-streaked into Tryptose Soy Broth Broth (Figure 9).
A control positive involved picking a loop full of Listeria monocytogenes
(Field stem – Laboratory of Veterinary Public Health, Faculty of Veterinary
Medicine- IPB) and mixing it with Listeria enrichment broth in a clean and sterile
erlenmeyer. A control negative involved the use of a loop full of Staphylococcus
aureus (ATCC 25923) with Listeria enrichment broth and both controls were run
in the same manner as the test samples.
Inoculation of Listeria monocytogenes in test media
Serial dilution of the culture was done with BPW (0.1% v/v) to achieve a
cell concentration of approximately 1.0 x 102 CFU/ml. One hundred milliliters
(100 ml) of sterile milk was used as a test sample in eight clean and sterile
erlenmeyer. Then a 0.1ml of it was aseptically pippeted into each of the seven
erlenmeyer with codes E1 to E7, but E8 was not inoculated because it was the
control sample. Then all the erlenmeyers were stored in the refrigerated incubator
set at 4oC. After 24 hours, erlenmeyer E1 was removed from the refrigerator
together with E8 as the control. Then 1ml of E1 was aseptically pipetted into

20

already prepared clean and sterile petri dishes with labels 100 (in duplicate) and
was also prepared using 9ml of BPW 10-1( in duplicate) , and then 1ml from E8
with labels KI and K2. This made a total of 6 petri dishes. Then each of the petri
dishes was flooded with a nutrient agar and mixed thoroughly in the form of
figure 8 and then left for some time to solidify before they were transferred to the
incubator that was set at 37oC and incubated for 24 to 48 hours. The control (E8)
was returned to the refrigerator while E1 was removed and never used again. The
next day, the colonies that grew were counted using a colony counter and the
results recorded, then followed up the next day (48hrs) and recounted to check for
any increment in the number of colonies, the results were also recorded. The
above procedures were used for the rest of the samples until all the 7 samples
were completed.

Figure 11 Milk samples for the quantitative analysis of Listeria monocytogenes
…………...kept in the refrigerator set at 4oC.
Data management and analysis
Program excel version 2003 (Microsoft Office excel 2003, Microsoft
Office Professional Edition, 2003) and Minitab 14 a statistical software were used
for collection, management and analysis of data. Descriptive statistics were used
to describe the results.

RESULTS AND DISCUSSION

Qualitative

21

There was no Listeria monocytogenes detected in the samples tested (Table1).
Table 4.

Presence of Listeria monocytogenes in pasteurized milk by the

……………qualitative method
Laboratory results
Supermarke
t code
G (n = 8)
H ( n = 8)
E ( n =8)
J ( n = 8)

Type of sample
AI –B1 –C1- D1A3 – B3-C3 – D3
AI –B1–C1- D1A3 – B3-C3 – D3
A2 –B2 –C2- D2A4 – B4-C4 – D4
A2 –B2 –C2- D2A4 – B4-C4– D4

Number of Positives
None

Positive (%)
0

None

0

None

0

None

0

Note: Letters G, H, E and J are codes of Supermarkets, A, B, C and D are codes of samples and 1, 2, 3 and 4
are weeks during which samples were collected from the Supermarkets.

Pasteurized milk is very much known to be one of the vehicles through
which Listeria monocytogenes is transferred into the human body. Because of
this, many countries have initiated a zero tolerance policy prohibiting the sale of
processed

ready-

to-

eat

food

products

contaminated

with

Listeria

moonocytogenes. This policy designates Listeria monocytogenes as an adulterant.
Despite this policy, Listeria monocytogenes is constantly found in these foods.
Vander-linde and Grav, (1991) found 2 (2.4%) Listeria spp. These numbers were
attributed to secondary contamination of different supplements and different
production methods. Dominguez et al.1985; Garcia and Vitas 2004 from Spain
found a much higher incidence of Listeria monocytogenes in pasteurized milk,
45.3 % and 44.7 % respectively. This was far much higher than what investigators
from other countries have reported. Gül et al. (1994) from Diyarbakir, Turkey,
reported 1.1 % Listeria monocytogenes in pasteurized milk samples.
In a related

study to develop data on the risk of listeriosis to support a

science-based strategy for addressing Listeria monocytogenes in foods in the
United States, eight categories of ready-to-eat foods were collected over 14 to 23
months from retail markets at Maryland and northern California FoodNet sites.
The product categories included luncheon meats, deli salads, fresh soft "Hispanicstyle" cheeses, bagged salads, blue-veined and soft mold-ripened cheeses, smoked

22

seafood, pasteurized milk and seafood salads. The presence and levels of L.
monocytogenes in the samples were determined by rapid DNA-based assays in
combination with culture methods. Of the 31,705 samples tested, 577 were
positive. The overall prevalence was 1.82%, with prevalence ranging from 0.17 to
4.7% among the product categories (Chen et al. 2003).
In Ankara, Turkey, Farber et al. (1988) reported (18.2%) of raw milk
contained Listeria monocytogenes. Sharif and Tunail (1991) tested pasteurized
milk samples and reported that all were negative for Listeria monocytogenes. This
research also found no sample positive to Listeria monocytogenes. The result is in
agreement with the findings of other researchers in other countries. Based on all
the findings mentioned above, it can be concluded that Listeria monocytogenes
has a very