Detection and Enumeration of Listeria spp. and Listeria monocytogenes in Fruit Juices

Chai Sze Fan

QR 201 L7 C434 Bachelor of Science with Honours 1013 (Resource Biotechnolo~)

2013

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Detection and Enumeration of Listeria spp. and Listeria monocytogenes in Fruit Juices p.KHIDMAT MAKLUMAT AKADEMIK

111111111 fli'~A~11111I1I1 1000246680

Cbai Sze Fan (26039)

A thesis submitted in partial fulfilment of the requirements for the degree of Bachelor of Science with Honours

Resource Biotechnology

Supervisor: Dr. Lesley Maurice Bilung Co-supervisor: Dr. Micky Vincent

! Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

2013

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,

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ACKNOWLEDGEMENT

This research project would not be able to be completed without the support of many

people. First of all, I would like to express my deepest gratitude to my supervisor, Dr.

Lesley Maurice Bilung who was helpful and has offered invaluable experience, assistance,

support and guidance throughout the project. Besides that, I would like to dedicate my

gratitude to my co-supervisor Dr. Micky Vincent for his valuable infonnation and

knowledge. Million thanks to post-graduate students of Microbiology Laboratory,

especially Miss Velnetti Linang, Miss Christy Chan Sien Wei and Mr. Yong Sy Fuh and

Mr. Ennry Ak Esut for sharing their methodology, experiences, tips and their willingness

in providing helpful assistance throughout the project. Also, special thanks to my beloved

laboratory colleagues for providing condusive working environment for these two

semesters and willing to lend their hand whenever I need help. Finally, I would like to r

thank my beloved families and friends for their patience, understanding and support

through all the hardships and joy of this project.

DECLARATION

I hereby declare that this thesis entitled "Detection and Enumeration of Listeria

monocytogenes in Fruit Juices" is based on my original lab work except for the citations

and references which have been duly acknowledged. Also, I declare that it has not been

previously submitted for any other degree of qualification or other institution.

Signature - Date

ii .....

Pusat Khidmat Maklumat Akadcmik UNIVERSm MALAYSIA SARAWAK

TABLE OF CONTENTS

PAGE

ACKNOWLEDGEMENT

DECLARATION 11

LIST OF ABBREVIATION V11

ABSTRACT

TABLE OF CONTENTS III

LIST OF TABLES AND FIGURES v

CHAPTER 1 INTRODUCTION 2

CHAPTER 2 LITERATURE REVIEW 6

2.1 Genus of Listeria 6

2.2 Listeria monocytogenes 7

2.2.1 Virulence factors 7

2.2.2 Characteristic important to food processors 8

2.2.3 Incidence of L. monocytogenes in fruit 9

2.2.4 Outbreak of Listeriosis 10

2.3 Most Probable Number (MPN) method 11

2.4 Polymerase Chain Reaction (PCR) 11

CHAPTER 3 MATERIALS AND METHODS 13

3.1 Sample collection and processing 13

3.2 Standard plate count 13

3.3 . Enumeration using MPN method 14

3.4 DNA extraction using boiled cell method 14

3.5 Detection using Polymerase Chain Reaction 15

1II

3.6 Gel electrophoresis 16

CHAPTER 4 RESULTS 18

CHAPTER 5 DISCUSSION 27

5.1 Most Probable Number (MPN) and Standard Plate Count 27

analysis

5.2 Specific Polymerase Chain Reaction (PCR) 28

5.3 The occurrence ofL. monocytogenes in fruit and fruit juices 29

CHAPTER 6 CONCLUSION 32

REFERENCES 33

iv

<!f-.

LIST OF TABLES

TABLE PAGE

2.2.3 Fruits or fruit products from which L. monocytogenes has been recovered 10

3.1.1 Fruit types and the number of fruit juices examined for the detection and 13

concentration of L. monocytogenes

3.5.1 Primers sequence used for the specific Polymerase Chain Reaction 15

3.5.2 List of components in PCR master mix 16

4.1 Results of MPN value, CFU value and PCR analysis of apple juice samples 21

4.2 Results of MPN value, CFU value and PCR analysis of orange j1!lice samples 22

4.3 Results of MPN value, CFU value and PCR analysis of watermelon juice

samples 23

4.4 Results of MPN value, CFU value and PCR analysis of honeydew juice

samples 24

4.5 Results ofMPN value, CFU value and PCR analysis of papaya juice

samples 25

v

1

LIST OF FIGURES

FIGURE PAGE

4.1 Gel electrophoresis image for apple, orange and honeydew juice sample 18

with L. monocytogenes positive control (ATCC 15313), hlyA gene

4.2 Gel electrophoresis image for honeydew and papaya juice sample with L. 19

monocytogenes positive control(ATCC 15313), hlyA gene

VI

bp

CFU

DNA

dNTPs

hlyA

kb

LLO

L. innocua

L. ivanovii

L. monocytogenes

L. seeligeri

MgClz

NCBI

ml

MPN

PCR

PC-PLC

PI-PLC

rpm

TBE

TNTC

TSA

TSB

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LIST OF ABBREVIATION

Base pair

Colony-forming Unit

Deoxyribonucleic acid

Deoxynucleotides triphosphate

Haemolysin gene

Kilobase

Listeriolysin 0

Listeria innocua

Listeria ivanovii

Listeria monocytogenes

Listeria seeligeri

Magnesium chloride

National Center for Biotechnology Information

Mililitre

Most Probable Number

Polymerase Chain Reaction

Phosphatidylcholine-specific phospholipase C

Phosphatidylinositol-specific phospholipase C

Revolution per minute

Tris-Borate-EDT A

Too numerous to count

Trypticase Soy Agar

Tryptic Soy Broth

Voltage

Microlitre

VII

V

Detection and Enumeration of Listeria spp. and Listeria monocytogenes in Fruit Juices

Chai Sze Fan

Resource Biotechnology Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

Listeria spp. is an important foodbome bacterial group that is able to cause food poisoning outbreak and diseases among human. In this research, five types of fruit juices were sampled to enumerate and detect the presence of Listeria spp. and Listeria monocytogenes (L. monocytogenes). The Most Probable Number (MPN) method was used for enumeration and specific Polymerase Chain Reaction (PCR) was performed targeting the virulence gene, hlyA gene with expected size of 730 bp to determine the presence of L. monocytogenes in the fruit juice samples. The hlyA gene confers haemolytic properties in L. monocytogenes as it produces Listeriolysin 0 (LLO) which involves actively in cell lysis process of phagocytic membrane. Out of the 50 fruit juice samples, all were negative for the presence of the hlyA gene. MPN analysis showed that the results of all samples showed estimated microbial load of Listeria species as more than 1100 MPN/g. For standard plate count, only three apple juice samples out of 10 apple juice samples showed range from 2 .8x 104 to 1.9x 108

CFUlml and only one honeydew juice sample out of 10 honeydew juice samples showed 4.0x 103 CFU/ml. Factors contributing to the absence of L. monocytogenes in the fruit juice samples were proper hygienic and sanitary production and processing practices applied by the workers in the fruit stalls and restaurants. Besides, low chances to get cross-contamination during fruit juices preparation and proper water supplies used in sampling sites also led to the absent of L. monocytogenes in fruit juice samples.

Keywords: L.monocytogenes, fruit juices, MPN, standard plate count, peR, hlyA gene

ABSTRAK

Spesies Listeria merupakan sejenis kumpulan bakteria bawaan makanan yang dapat menyebabkan makanan wabak keracunan dan penyakit di kalangan manusia. Dalam kajian ini, lima jenis jus buah-buahan telah disampel un/uk menghitung dan menges an kehadiran spesies Listeria dan L. monocytogenes. Kaedah Bilangan Paling Mungkin (MPN) telah digunakan untuk penghitungan dan Tindak balas rantai polymerase (PCR) spesijik lelah dijalankan untuk mengesan gen virulens, gen hlyA dengan saiz jangkaan 730 bp, yang dapat menentukan kehadiran L. monocytogenes dalam sampeljus buah-buahan. Gen hlyA memberikan sifat hemolitik kepada L. monocytogenes dengan menghasilkan Listeriolysin a (LLO) yang terlibat secara aktif di dalam proses sel /isis di membran berjagosit. 50 sampel jus buah-buahan menunjukkan keputusan negati/ dalam penggesahan gen hlyA. Manakala bagi analisis MPN, keputusan semua sampel menunjukkan anggaran mikrob lebih daripada 1100 MPNIg. Sebaliknya, untuk kiraan plat standard, hanya tiga sampel jus epal daripada 10 sampel jus epal menunjukkan pelbagai daripada 2.8x 104 to 1.9x 108 CFVlml dan hanya satu jus tembikai susu sampel daripada 10 sampel jus tembikai susu menunjukkan 4.0 x ld CFVlml. Faktor yang menyebabkan L. monocylogenes lidak dapal dikes an di dalam sampel jus buah-buahan adalah pengamalan kebersihan, pengeluaran sanitari dan amalan pemprosesan yang betul yang diamalkan oleh pekerja di gerai buah-buahan dan resloran. Selain ilu, peluang yang rendah untuk mendapatkan kontaminasi bersilang semasa penyediaan jus buah-buahan dan pembekalan air yang betul yang digunakan di tapak persampelan juga menyebabkan L. monbcytogenes lidak dapat dikesan di dalam sampel jus buah-buahan.

CHAPTERl

INTRODUCTION

Fruit is an essential component for a healthy diet. It consists of high concentration of

vitamins, minerals and fibres. Fibres provided in the fruit can ward off cholesterol and fat

from the body and prevent constipation from occurring. Vitamins found are involved in

boosting human immune system (Hoffman, 2004). Moreover, fruits also contain variety of

phytonutrients which able to act as anti-oxidants and protect human body from harmful free

radicals and chronic diseases such as cancer and heart diseases (British Broadcasting

Corporation (BBC), 2013). According to British Broadcasting Corporation (2013), population

studies have shown that people who consume a lot of fruits may have lower risk of

developing chronic diseases. Fruits can be processed into quite a number of products such as

sweet, salad and fruit juices. Among the products, fresh fruit juices are preferred by most

consumers as they are minimally-processed convenient foods that are ready-to-eat. Due to

this reason, there is a significant rise of consumption rate of fresh fruit and changes in

consumption trend among human population (Abadias et aI. , 2008).

Although fruit is highly consumed for its health benefits, it still possesses some

potential risks. Fresh fruits are natural carrier of non-pathogenic epiphytic microflora.

Furthennore, fruits can be contaminated with pathogens during growth, harvest,

transportation and further downstream process (European Commission Health and Consumer

Protection Directorate-General, 2002). Normally, fresh fruit juices are prepared by extracting

or mechanical means and have no or little steps taken to reduce contamination and pathogen

level (Victorian Government Department of Human Services, 2005). Contamination from

raw fruits, equipment of food handlers can easily be transmitted to final product during the

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preparation process and causes microbial contamination of final product can occurred.

Moreover, fresh fruit is an ideal place to habour foodborne pathogen such as Listeria spp. and

it is also a vehicle of transmission of foodborne diseases into human body (Bui et ai., 2012).

Hence, chances of getting food poisoning are always there.

Listeria monocytogenes (L. monocytogenes) is a Gram positive, facultatively

anaerobic non-spore forming and rod-shaped bacteria (Jeyaletchumi et ai., 2010). L.

monocytogenes is one of the most important causes of human food borne infection worldwide.

It is able to cause serious disease called listeriosis. Jadhav et al. (2012) stated that, listeriosis

may cause abortions and premature deliveries during birth, lead to septicemia and meningitis

among neonates and causes meningitis, encephalitis, meningoencephalitis and septicemia to

immunocompromised individual. According to the World Health Organization (2004), the

incidence of listeriosis is relatively rare as compared with other foodborne diseases such as

typhoid fever, but it leads to high fatality rate (20-30%).

The interest in this pathogen arise after several disease outbreaks in the early 1980s

(0' Connor, 2002). The most recent outbreak of listeriosis was in 2011 and 2012. In 2011,

the outbreak of listeriosis related to whole cantaloupe from Jensen Farms has occurred in the

United States and caused 33 deaths in total (Centre for Disease Control and Prevention, 2011).

In 2012, the outbreak was originated from the imported Frescolina Marte Brand Ricotta

Salate Cheese in 12 states in United States and the District of Columbia. This outbreak has

infected 20 persons (Centre for Disease Control and Prevention, 2012). In Malaysia, the

presence of L. monocytogenes were detected in various food samples including raw and

ready-to-eat foods such as vegetarian burger patties, salad, vegetables, chicken and egg

products (Wong et ai., 2011 ; Jamali et aI., 2012; Marian et aI., 2012).

3

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L. monocytogenes is normally found in food processing and farm environment such as

in soil, water, sewage and vegetation. It is able to survive under high salinity, acidic condition,

refrigeration temperatures and low water activity (Jadhav et aI., 2012). However, the

organism will not survive under pasteurization temperatures. Pathogenic contamination may

occur on fresh fruits during growing, harvesting, handling or distribution state. Hence, it is a

possible to detect the presence of L. monocytogenes in unpasteurised or untreated fruit juices

during juicing process (NSW Food Authority, 2009). Besides that, Brackett (2007) mentioned

that the presence of L. monocytogenes in fruit juices may also be due to post-pasteurization

contamination where L. monocytogenes contaminated raw milk is introduced into fresh juices

during fruit juices preparation. In Malaysia, the less acidic tropical fruits such as papaya,

watermelon and banana favour the growth of L. monocytogenes (Brackett, 2007).

The Most Probable Number (MPN) method provides quantitative data on the

concentration of viable L. monocytogenes in the fruit juice sample (Ubong et aI. , 2011). The

result relies on the turbidity and gas formation in MPN broth tube. MPN method shows

advantages even when the contamination is low. On the other hand, Colony-forming unit

(CFU) is the estimation of the number of viable cells present based on the colonies formed.

The method is based on the ability of L. monocytogenes to give rise to colonies under

provided nutrient medium. The sample will be diluted until the bacteria colonies can be

counted on the plate and the ideal plate count should be ranging from 30 to 300 colonies on a

standard sized petri dish (Reynolds and Farinha, 2005).

In this study, Polymerase Chain Reaction (PCR) was used to amplify the haemolysin

gene (hlyA) of L. monocytogenes present in the fresh juice samples (Jeyaletchumi et aI.,

2010). HlyA gene is a virulent gene which codes for Listeriolysin 0 (LLO) that is responsible

to promote haemolytic activity. The gene is located in the PrfA-dependent virulence gene

cluster. According to Kuhn and Goebel (2007), the haemolytic activity detected around

4

Pusat Khidmat MakJumat Akademik UNIVERSm MALAYSIA SARAWAK

colonies of 1. monocytogenes on blood agar is a major virulence determinant because all

clinical isolates of L. monocytogenes show this haemolytic phenotype. Moreover, Kuhn and

Goebel (2007) also stated that in experimental infection, all virulent strains were found to be

haemolytic while non-haemolytic strains were avirulent. Hence, hlyA gene can be used as one

of the determinant to detect the presence of L. monocytogenes.

In Peninsular Malaysia, previous researches have reported the presence of Salmonella

spp., Salmonella typhi and Salmonella typhimurium in fruit juices (Diana et aI. , 2012). There

is yet a study carried out in East Malaysia on the distribution of L. monocytogenes in fruit

juices. Hence, the findings from this study will provide baseline data on L. monocytogenes

contamination in fruit juices, as well as an insight of the potential risk for fruit juices

consumption in Malaysia. Therefore, the present study was designed to meet the following

objectives:

1. To enumerate Listeria spp. and L. monocytogenes in fruit juices using MPN method

and standard plate count.

2. To detect the presence of L. monocytogenes in fruit juices by using peR assay,

targeting hlyA gene.

5

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CHAPTER 2

LITERATURE REVIEW

2.1. Genus of Listeria

The genus of Listeria contain only L. monocytogenes during its first discovery. In

1948, Listeria denitrificans (L. denitrificans) was added to the group but it is later been

transferred to the genus of Jonesia, which belongs to the corynefonn group of bacteria

(Rocourt and Buchrieser, 2007). In 1966, Listeria grayi (L. grayi) was added followed by

Listeria murrayi (L. murrayi) in 1971. H]owever, through the numerical taxonomic,

DNAIDNA hybridization and mutlilocus enzyme electrophoresis activity conducted, the

result showed that L. murrayi should belong to a single species: L. grayi (Stuart and

Welshimer, 1974). Listeria innocua (L. innocua) was added as one of the member in 1981

followed by Listeria welshimeri (L. welshimeri) and Listeria seeligeri (L. seeligeri) in 1983

and lastly, Listeria ivanovii (L. ivanovii) was added in 1985. Hence, the present listeria genus

now consists of six species, L. monocytogenes, L. ivanovii, L. seeligeri, L. innocua, L.

welshimeri and L. grayi. Among the six species of Listeria, only L. monocytogenes and L.

ivanovii are pathogenic species where L. monocytogenes is the primary human pathogen that

causes listeriosis and able to infect other warm-blooded host species such as ruminant.

(Bakker et al. 201 0). On the other hand, L. innocua, L. seeligeri, L. welshimeri and L. gravyi

are non-pathogenic species. Schmid et al. (2005) pointed out that, L. gravyi has a low

relatedness with other five species of Listeria and is representing a different genus, Murraya.

According to Bakker et af. (2010), pathogenic species is closely related to non-pathogenic

species in term of 16S- rRNA; L. monocytogenes is closely related to L. innocua and L.

welshimeri, on the other hand L. ivanovii is closely related to L. seeligeri.

6

Listeria is a Gram positive small bacterium with 0.5 11m in diameter and 1 to 2 11m in

length, rod shaped with rounded end (Rapeanu et af., 2008). It is a facultative anaerobic, non­

spore forming bacterium. Listeria is nonnally found in single unit or in short chains, and

sometimes may arranged in V and Y fonns or in palisades (Rocourt and Buchrieser, 2007).

Motility of Listeria depends on its few peritrichous flagella and shows tumbling motility

where it twists and wriggles to produce fast and eccentric rotations before it suddenly move

quickly in various directions (Rocourt and Buchrieser, 2007). The growth rate of Listeria in

media is facilitated by the presence of fennentable sugar such as glucose. On plate culture, it

will produce acid odor due to the fonnation of carboxylic acids, hydroxyl acids and alcohol

(Daneshvar et al., 1989). While in broth, turbidity can be observed after 8 to 24 hours of

incubation at 37°C with pH between 4.5 and 9.2, optimally at pH 9. According to Rocourt

and Buchrieser (2007), the temperature limits of growth rate of Listeria are I-2°C to 45°C.

The temperature strongly affects the survival of Listeria at low pH and high salt concentration.

This organism is able to grow in 10% NaCI and survive at higher concentrations.

2.2 Listeria monocytogenes

2.2.1 Virulence factors

L. monoc:ytogenes contains 13 serovars, but only three serovars which are 112a, 1I2b

and 4b are responsible for more than 90% of the diseases (Janzten et af., 2006). Among these

three serovars, serotype 4b is the most common infectious agent. L. monocytogenes has the

ability to induce its own uptake into phagocytic and non-phagocytic cells. It enters the human

body through the intestine when contaminated food is consumed. Virulence genes are

clustered in the Prf-dependent virulence gene cluster which comprised of six virulence genes:

prfA, picA, hly, mpl, actA and picE (Kuhn and Goebel, 2007). The products of these virulence

7

genes are listeriolysin (LLO) encoded by hly, a phosphatidylinositol-specific phopholipase C

(pL-PLC) encoded by plcB, a metalloprotease (Mpl) encoded by mpl, ActA protein encoded

by actA and the positive regulatory factor PrfA which encoded by prfA. On the other hand,

intemalin A (InlA) and internal in B (lnlB) are encoded by inlAB operon. The invasion of

non-phagocytic cells is facilitated by the internal in protein A and B, which mediate adhesion

and invasion of host cells, found on the surface of listerial cells (Doyle, 2001).

After the uptake by mammalian cells, L. monoc;ytogenes is able to escape from the

vacuole through the action of LLO which causes haemolytic activity. Haemolysin is the

major virulence factor of L. monocytogenes. According to Kuhn and Goebel (2007), the

haemolytic activity caused by the action of LLO which is detected around the colonies of L.

monocytogenes on blood agar is a major virulence determinant because all clinical isolates of

L. monocytogenes show this haemolytic phenotype. LLO belongs to a family of thio-activated,

cholesterol-dependent, pore-forming toxin which is secreted to lyze the vacuolar membrane

and escape into the cytoplasm (Kuhn and Goebel, 2007). The spreading action from cell to

cell is completed by using actin-motility process. The actin filament is formed through the

induction of poLymerization of globular actin molecule by listerial cell surface ActA protein

(Doyle, 2001). Furthermore, L. monocytogenes is capable to synthesize phosphatidylinositol­

specific phospholipase C (PI-PLC) and phosphatidylcholine-specific phospholipase C (PC­

PLC) which take part in the escape of L. monocytogenes from phagocytic vacuole (Hii, 2009).

2.2.2 Characteristic important to food processors

Lado and Y ousef (2007) stated that L. monocytogenes is able to grow at temperature

between -1.5 and 45°C and has an optimal growth temperature between 30 and 37 °C. The

bacterium will not survive under pasteurization temperatures. Heating L. monocytogenes at

8

temperature above 56°C will cause ribosomal damage, protein denaturation and enzyme

inactivation (Anderson et al., 1991). Besides that, the ability of L. monocytogenes to grow in

refrigeration temperature is due to the presence of more than 95% of branched-chain fatty

acids in the membrane phospholipid of L. monocytogenes which help to retain the membrane

phospholipids in liquid-crystalline state and therefore maintain the membrane fluidity and

growth during low temperature (Lado and Y ousef, 2007). The optimal growth pH for L.

monocytogenes is close to neutrality, pH 7, ranging from 4.1 to 9.6 (Lado, 2003). The effect

of pH on viability of L. monocytogenes also depends on other environmental factors such as

temperature. Meanwhile, the ability of L. monocytogenes to maintain an intracellular pH

close to neutrality, regardless of environmental pH, is the key point to maintain cell's

viability. When a threshold pH is reached, the cell will cease to expel proton fast enough and

cause a decrease in intracellular pH (O'Driscoll et aI., 1997). Furthermore, according to Lado

and Yousef (2007), L. monocytogenes is able to survive with relatively low water activity, as

low as 0.90. In additon, L. monocytogenes is able to tolerate extremely high salt concentration,

survive at refrigeration and frozen temperature (Food and Drug Administration, 2008). For

example, the bacterium is able to survive in commercial cheese brine with 23.8% of sodium

chloride, pH 4.9 stored at 4 °C for 259 days (Lado and Y ousef, 2007).

2.2.3 Incidence of L. monocytogenes in fruit

According to Brackett (2007), when compared to the availability of information on

the growth and survival of L. monocytogenes in vegetables, insufficient information on the

incidence of L. monocytogenes in raw fruits or fruit products are available. The low incidence

of li~teriae on fruits may be due to the fact that most fruits are grown on the tree or above the

ground and therefore there is less frequent contact with L. monocytogenes-contaminated soil,

9

water or faeces (Brackett, 2007). Furthermore, tropical fruits are often less acidic, hence

greater likelihood for survival and growth of L. monocytogenes could be expected on tropical

&Oits as acidic pH is unfavorable pH for L. monocytogenes to grow and survive (Brackett,

2007). Cantaloupe from Jensen Farms in Colorado is the first whole fruit reported to be

contaminated by L. monocytogenes (Food and Drug Administration, 2011). Table 1 shows

the fruits or fruit products from which L. monocytogenes has been recovered.

Table 2.2.3: Fruits or fruit products from which L. monocytogenes has been recovered (Brackett, 2007)

Country Food United States Apple, blueberries, cantaloupe, dried fruit, fruit salad, fruit products,

melon, pears, pineapple and watermelon. Czech Republic Dried fruit

Germany Fresh fruit and fruit product

I'

2.2.4 Outbreak of Listeriosis

In 1981, outbreak of listeriosis in epidemic proportion was recorded in the Maritime

Provinces of Canada and over 100 people were involved (Tador, 2009). This outbreak has

rose the public awareness toward the foodbome transmission ofL. monocytogenes to humans.

The multistate outbreak of listerosis caused by whole cantaloupe and imported Frescolina

Marte Brand Ricotta Salate Cheese in 2011 and 2012 is the most recent outbreak caused by L.

monocytogenes. The pathogen was isolated from the sample of cantaloupes from Jesen farms

in Coloroda in 2011 (Centers for Disease Control and Prevention, 2011). In October 2012,

20 listeriosis infected people has been reported from 12 states in United State and the District

ofColumbia and indicated that imported Frescolina Marte Brand Ricotta Salate Cheese is the

source ~fthe outbreak (Centers for Disease Control and Prevention, 2012).

10

2.3 Most Probable Number (MPN) method

The MPN method is a method used to obtain quantitative data on the concentration of

viable microorganisms in a sample. It is useful to detect low level of microorganisms in

Jiquid samples. One of the biggest advantage of this method is that only viable

microorganisms are enumerated (Food and Drug Administration, 2010). MPN method is

accompanied with serial dilution test where usually ten-fold dilutions are perfonned in either

3, 5 or 10 tubes MPN series. The result is based on the observed positive growth response

such as turbidity and gas fonnation in broth tubes (United State Department of Agriculture,

2008). The number of positive tubes at each dilution will suggest an estimation of the original,

undiluted concentration of microorganism in the sample (Food and Drug Administration,

2010). Theoretically, higher dilution's sample will obtain fewer positive tubes in the series.

United State Department of Agriculture (2008) stated that few assumptions should be

included during the test: the microorganisms are randomly and evenly distributed in the

sample, the microorganisms is not clustered and repelling each other and every tube which

may only contains single viable microorganism is able to produce positive response.

2.3 Polymerase Chain Reaction (peR)

Polymerase Chain Reaction (PCR) is a technique used to amplify DNA sequences of

targeted organisms. It requires one set of primers that specifically bind to the DNA sequences

at opposite ends of opposing strand of DNA, a thermostable DNA polymerase (usually Taq

DNA polymerase is used), double stranded DNA, in this case pathogen-specific target DNA

is used, four type of deoxynucleotides triphosphate (dNTPs), PCR buffer and magnesium

chloride (MgCh) in order to complete the reaction (Jeyaletchumi et at., 2010). It involves

three simple steps, namely denaturation, annealing and elongation. The amplified product is

11

ide1:ectc~d through gel electrophoresis combined with ethidium bromide staining and visual

'·~A''''II'II''~'V'' of the gel using UV light (0' Connor, 2002). The amplified target DNA is

_ lAnllfpn by applying electric current, according to its size and charge. Although there are

other nucleic acid detection approaches, PCR is still remains the most widely applied

(Jeyaletchumi et aI., 2010). Diana et al. (2011) reported the detection of

Salmonella spp., Salmonella typhi and Samonella typhimurium in fruit juices using PCR

assay. Besides that, PCR assay also has been applied in the detection of L. monocytogenes in

vegetarian burger patties in Malaysia (Wong et aI., 2011).

12

CHAPTER 3

MATERIALS AND METHODS

Sample collection and processing

Fifty fresh fruit juices were collected randomly from fruit juice stalls and restaurants

located in the Kota amarahan and Kuching, Sarawak. The fruit types and the number of fruit

juices examined for the detection and concentration of L. monocytogenes was shown in Table

3.1.1. The fruit juice samples were placed in an ice box immediately after collection and

processed at the Microbiology Laboratory, UNIMAS.

Table 3.1.1: Fruit types and the number of fruit juices examined for the detection and concentration of L. monocytogenes

English name Scientific name Number of samples Orange Citrus sinensis 10 Papaya Carica papaya 10

Watermelon Citrullus lanatus 10 Honeydew Cucumis melo 10

Apple Malus domestica 10 Total: 50

Sample processing was performed as described by Wong et al. (2011). Ten mililitre of each

sample was transferred into 90 ml of Tryptic Soy Broth (TSB) (Becton, Dickinson &

Company, France). The samples were pre-enriched and placed in an incubater shaker at 37°C

for 24 hours .

Standard plate count

Standard plate count was performed as according to Reynolds and Farinha (2005)

with minor modification. Enriched samples were diluted with ten-fold serial dilution. One

13

.2

.3

ofeach sample was transferred to 9 ml of saline in first test tube to fonn 10-1dilution.

one mililitre of diluted sample was transferred from 10-1 test tube into second test tube

10-2 dilution. The step was repeated with higher dilution factor. One hundred

of each dilution was directly plated onto PALCAM agar (Merck KGaA Dannstadt,

~nmUlY). The agar plate was incubated at 37°C for 24 to 48 hours. After that, the number of

:COI,on~'-t(Jlrm,mg units were counted and recorded. Only petri plate containing between 30 and

colonies was selected. The number of bacteria (CFU) per millilitre of fruit juices was

;CUCUIl:lte<1 by using the fonnula below:

number of colonies = number of bacteria (cfu/ml) dilution factor x volume plated

(Source: Reynolds and Farinha, 2005)

Enumeration using MPN method

Enumeration of L. monocytogenes was perfonned as described by Wong et al. (2011).

One mililitre of three successive ten-fold dilutions were transferred into three test tube set

eontaining 9 ml of Fraser broth base (OXOID Ltd, England). Fraser broth base (OXOID Ltd,

~EIltgiand) contains lithium chloride and aesculin which has an advantage for detecting Listeria.

tubes were incubated at 37°C for 18 to 24 hours. Turbid MPN tubes were identified and

number of turbid tube was recorded.

DNA extraction using boiled ceU method

L. monocytogenes DNA was extracted using the boil cell method as according to

UJIIIU&' C" al. (2011). One mililitre of sample of each broth was centrifuged (Hettich EBA 21

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_ftoiti.n"orI

Germany) at 12,000 rpm for 3 minutes to pellet the cellular debris. The

lUPemataJnt was discarded and the cell pellet was resuspend in 200 III of sterile distilled water

was vortexed (Labnet International, USA). Then, the ceU suspension was boiled for 15

IbiIlutf~ and immediately cooled at -20 DC for 15 minutes. The cell suspension was then

(Hettich EBA 21 Zentrifugen, Germany) for 3 minutes at 12,000 rpm. Five

_u........rr".ofthe supernatant as DNA template solution was used as template for PCR assay.

Detection using Polymerase Chain Reaction

PCR assay was performed according to Kargar and Ghasemi (2009) with minor

iD'lClldll:lcatlon in the concentration of reagents and primer optimization. One set of primers as

\1R1I1D11l~anzeo in Table 3.5.1 were used to detect hlyA gene in L. monocytogenes isolates.

Table 3.5.1: Primers sequence used for specific Polymerase Chain Reaction

Primer Primer sites (5' - 3') Amplicon sizes (bp)

hlyA-F CAT TAG TGG AAA GAT GGA 730 bp (Gouws and ATG Liedemann, 2005)

hlyA-R GTA TCC TCC AGA GTG ATC GA

Twenty-five microlitre of PCR reaction was performed. The reagents needed including lOX

buffer (Fermentas International Inc, Canada), 25 mM of Magnesium Chloride (MgCb)

@,cmnent!S International Inc, Canada), 10 mM dNTP mix (Fermentas International Inc,

·_IIIUClIj, 10 mM hlyA-F and hlyA-R primers (Fermentas International Inc, Canada), 10 mM

DNA polymerase (Fermentas International Inc, Canada) and sterile distilled water

The volume of each PCR reagents needed were shown in Table 3.5.2. Before

.lDUlg the PCR, positive and negative control were prepared. The positive control' contained

Jll of DNA template ATCC 15313 (American Type Culture Collection (A TCC), USA),

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