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
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
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.
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.
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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
Tittle of Thesis : Detection of Listeria monocytogenes in Pasteurized Milk Sold in Supermarkets in Bogor City and its Relationship with Human Health
.
Name : Robert Kibuuka
Registration number : B251078041
Study Programme : Veterinary Public Health
approved by supervisors
Prof.Dr.Drh. Hj. Mirnawati Sudarwanto Dr. Drh. A.Winny Sanjaya, MS
Head Member
acknowledged by
Head of Study Program Dean of Graduate School
Dr. Drh. Denny W. Lukman, MSi Prof. Dr. Ir. Khairil A. Notodiputro, MS
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:
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
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.
i
Listeria monocytogenes and the Disease ... 4
Growth of Listeria monocytogenes in Cold Storage ... 5
Sources of Listeria monocytogenes in Retail Shops ... 6
Listeria monocytogenes and its Characteristics ... 8
Occurence and Ecology of Listeria monocytogenes ... 11
The Disease in Man ... 13
Control of Listeria monocytogenes in Foods... 14
Chromogenic Medium for Isolation of Listeria monocytogenes ... 15
III. MATERIALS AND METHOD
Qualitative Analysis of Listeria monocytogenes... 18
Innoculation of Listeria monocytogenes in Sterile Milk... 19
ii
LIST OF TABLES
Page
1 Biochemical Differentiation of Listeria species... 9
2 Counts of Listeria monocytogenes in Milk at 4oC for 7 days... 10
3 Listeria moncytogenes Inactivation Temperature... 10
4 Results of Detecting the Presence of Listeria monocytogenes in Milk... 23
iii
LIST OF FIGURES
Page 1 Scanning Electron Micrograph of Listeria monocytogenes... 5 2 Consumers Carry the bacteria on Clothes while Shopping... 6 3 Schematic Representation of a Milk Suply Chain... 7 4 Listeria monocytogenes Gram Staining... 8 5 Growth and Valiability of Listeria monocytogenes in Certain
Foods at Freezing ... 10 6 Ways in which Listeria monocytogenes is Disseminated in the
Environment, Animals and Humans... 11 7 Listeria monocytogenes Pathways into the Food Processing Plant... 13 8 Morphology of Listeria species Growth on ALOA Agar Plates... 15 9 Flow Diagram for the Qualitative Isolation of Listeria
monocytogenes Foods... 18 10 Milk Samples for Quantitative Analysis of Listeria monocytogenes
kept in Refrigerated Incubator set at 4oC... 22 11 Regression Curve for Growth of Listeria monocytogenes in Sterile
iv
LIST OF APPENDICES
Page 1 Sampling Plan... 35 2 List of Abbreviations... 35 3 Flow Diagram for Qualitative Analysis of Listeria monocytogenes
growth in an Innoculated Milk sample in Nutrient Agar... 36 4 Regression Analysis for Listeria monocytogenes growth in Sterile
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 (aw) 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
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 (>104/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
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 value-added products in developing countries are strategic keys for future economic development. This will require the implementation of reliable in-plant HACCP-based 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 intra -regionally and internationally, locking many farmers out of a chance to improve their economic well-being. refrigerator at 4oC and relate it to the health of the consumer.
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.
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
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
7
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).
Raw milk receiving
Filter
Raw milk storage
Clarifier or Separator
Pasteurization
Filler
Cold storage
Distribution
Packaging material Vitamin addition Homogenized
Raw cream storage
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).
9
Table 1 Biochemical differentiation of Listeria species (FDA 2003)
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 cfu/ml
0 0
I 0
II 40
III 150
IV 800
V 2500
Table 3 Listeria monocytgenes inactivation temperatures (FDA 2003)
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.
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
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
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
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
Figure 8 Typical morphology of Listeria Spp. growth on ALOA agar plates (Anonymous 2001).
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 4oC 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 7oC. 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
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
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.
Figure 9 Qualitative Isolation of Listeria monocytogenes LEB (100ml)
Oxford (24 - 48 hours), 37 oC
TSAYE
(incubation for 24 hours, 37 oC
KOH test
TSB YE
Sugar
(Mannitol, Xylose, Rhamnose)
SIM Milk Sample
(10 ml)
Mix
Incubation 300C, 24,48hrs,
until 7 days and Streak
After growth, pick 5 suspected black colonies
(black
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
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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
21
There was no Listeria monocytogenes detected in the samples tested (Table1).
Table 4. Presence of Listeria monocytogenes in pasteurized milk by the 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.
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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 low occurrence in foods. However, in this research, this does not mean that all the samples were free of Listeria monocytogenes, maybe other factors may have played a big role.
Mesophilic aerobic bacteria and lactobacilla overwhelming flora that were seen may prevent proliferation of Listeria. Some authors reported that Lactobacillus sake (Lb.706) and similar spp, produce bacteriosin and play role in inhibiting proliferation of Listeria spp (Weis 1989; Johnson et al. 1989). Stopforth et al. (2005) reported that L. monocytogenes does not proliferate in food with high total microorganism numbers. Inhibitory effect on the growth of L. monocytogenes is mainly related to the microbiological composition of the raw milk, in terms of thermophilic Lactobacillus and yeast. Inhibition of L. monocytogenes is probably due to the interrelationship between microbiological and chemical factors.
Besides this the control of L. monocytogenes in pasteurized milk via HACCP is focused on the selection of raw milk and the control of the processing, packaging, distribution and storage conditions. Although the pathogen is effectively controlled during pasteurization, its presence in the finished product is possible as a result of post-pasteurization contamination from sources in the plant environment. At present, however, it is presumed that L. monocytogenes is killed by heating to 72°C at least 15 seconds.
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Growth of inoculated Listeria.monocytogenes in a commercial sterile whole milk product was monitored at 4°C for a period of one week and the results obtained were tabulated and a growth curve drawn (Figure12).
No. of days under storage at 4 (degree Celcius)
L
Figure 12 Growth curve for Listeria monocytogenes in commercial sterile milk stored in the refrigerator at 4°C for 7 days.
Table 5 Total Colony counts for Listeria monocytogenes in commercial …...sterile milk stored in the refrigerator at 4°C for 7 days.
Listeria monocytogenes maintained its population relatively well in sterile milk stored at 4°C as seen in figure 12. This is because it is a psychrotrophic
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organism which grows at lower temperatures. On the first, second, third and fourth day there was no significant increase in the number of colony forming units. However, on the fifth day a dramatic increase in the number of colony forming units was observed. On the sixth day, a slight decrease in colony forming units was noted if compared with the fifth day. However on the seventh day, the population of colony forming units shot up. From the growth curve it can be noted that increase in number days has a strong relationship with the increase in number of Listeria monocytogenes. Delignette – Muller et al. (2005) reported a linear relationship between and growth of Listeria monocytogenes. The reasoning for this kind of graph is likely an initial shock to the cells which may make them harder to recover.
Baranyi et al. (1995) reported that the predictions for Brochothrix thermosphacta growth were good when the temperature profile contained step changes from 17 – 25oC down to 5oC, but the predictions no longer held for steps changes down to 4oC. In this study the author also attributes this observation to the alteration of the physiological state of the organism caused by the sudden cold – shock which may result in additional lag phase. This initial shock may also be due to the lack of growth compounds in the media associated with these results. The cells become easier to recover as they adapt to the environment at the later stages. This model provides the dairy industry with a useful tool for effective management and optimization of product safety and can lead to more realistic estimations of pasteurized-milk related safety risks. It can therefore be presumed that Listeria monocytogenes concentrations on the fifth, sixth and seventh day of milk storage in the refrigerator at 4oC can be quite dangerous to human health in both the immune-compromised and the healthy individuals. A population of 102cells is already enough to cause serious listeriosis in the non- healthy individuals (Invasive form) and in healthy individuals (non – invasive form) since it requires just over 105 cells.
Doyle et al. (2001) reported that L. monocytogenes grows in pasteurized milk, with the numbers increasing 10-fold in 7 days at 4oC. Therefore, fluid milk
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week. Therefore temperature abuse may further enhance the multiplication of bacterial cells. In relation to the above condition, Hayes (1996) noted that the organism achieved a significant increase on the fourth and the fifth day while this research obtained a drastic increase from the fifth day, the numbers increasing unreasonably high on the sixth day. However, on the seventh day there was no difference in the number cfus if compared with those counted on the sixth day. This could have been due to exhaustion of nutrients in the media and also lack of space for multiplication. Therefore, the public health importance and the sanitary measures for the control of the organism in ready – to – eat foods that have longer shelf - lives and stored under refrigeration is very much important.
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All the samples collected from the four different Supermarkets in Bogor City were negative to Listeria monocytogenes based on the FDA method for the isolation and detection of Listeria monocytogenes in foods. This means that the results are in agreement with hypothesis which states that there is no Listeria monocytogenes in pasteurized milk sold in Bogor City because of the effectiveness of the pasteurization process and the fact that there is no post - pasteurization contamination. The risk assessment reinforces past conclusions that food borne listeriosis is a moderately rare although it can lead to severe disease.
Listeria monocytogenes is capable of resisting cold temperatures, that is to say the 4oC cannot inactivate the organism, though other organisms cannot survive. This model also illustrates that L. monocytogenes can multiply to very high numbers following storage under this temperature.
Recommendation
A survey should be done on samples from traditional markets to identify L.monocytogenes. Other available new methods with a much higher sensitivity like PCR together with rapid tests should be used. Every effort should be made to ensure that ready - to- eat foods with longer shelf – life are free of Listeria monocytogenes since few cells can multiply to very high numbers under refrigeration temperature.
The results of this assessment will assist government in the evaluation of the adequacy and focus of current programs, help in the development of new programs to ensure that these programs protect the public health and to evaluate the effectiveness of new strategies to minimize the public health impact of food-borne listeria.
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ALOA = Agar Listeria according to Ottaviani and Agosti CAMP = Christie – Atkins – Munch – Petersen test.
CFU = Colony – Forming Units LEB = Listeria enrichment broth FDA = Food and Drug Administration
ISO = International Organization of Standardization
LMBA = Listeria monocytogenes Blood Agar
PALCAM = Selective solid media containing Polymyxin B, Acriflavin, Lithium chloride, Ceftazidine, Aesceculin and Mannitol RTE = Ready to Eat foods
TSBYE = Tryptic Soya broth with yeast extract HACCP = Hazard Analysis and Critical Control Point USA = United States of America
HIV/AIDS = Human Immune Virus/ Acquired Immunodeficiency Syndrome
Appendix 3. Schematic flow chart for quantitative analysis of
………L.monocytogenes growth in contaminated sterile milk
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ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml
Pippete 0.1 ml
E1 E1 E1 E1 E1 E1 E1 E8
E1 E1……….E8
10-1 10-2 10…
Appendix 4: Regression Analysis: Colony counts versus No. of days under
………...refrigeration
BPW 0.1
%
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The regression equation is
Colony counts (cfu/ml) = 1.78 + 0.673 No. of days under storage at 4oC
Predictor Coef SE Coef T P Constant 1.7831 0.6957 2.56 0.050
S = 0.823203 R-Sq = 78.9% R-Sq (adj) = 74.7%
Analysis of Variance
Source DF SS MS F P Regression 1 12.687 12.687 18.72 0.008 Residual Error 5 3.388 0.678
