ISOLATION AND DETECTION OF CAMPYLOBACTER

JEJUNI AND BACILLUS CEREUS IN RETAIL FOOD

SAMPLES BY POLYMERASE CHAIN REACTION

ABSTRACT

One of the concerns in food industry is the contamination by pathogens, which is

a frequent cause of food borne diseases. Over the past decade, recurrent

outbreaks of diarrhoea worldwide, occurred by foodborne pathogen

Campylobacter contributed to its status as hazard. Among the food borne

pathogens, Campylobacter jejuni and spore forming Bacillus cereus are

considered to be the most food poisoning agents. In the present study, C. jejuni

and B. cereus were isolated from raw and processed poultry chicken including

breast and thigh among which only raw chicken samples showed the presence of

these bacteria. These isolated bacterial strains were then identified by using

conventional methods including biochemical testing such as catalase, oxidase. C.

jejuni was found causing sodium hippurate hydrolysis and B. cereus was

identified by using API 50CH. These bacterial strains were further subjected to

PCR for identification where DNA was extracted from both of the samples and

DNA extraction of both the bacterial strains was successful but the DNA band

size obtained was less than that expected. The sequences generated by PCR

amplification with specific sets of oligo primers were unidentified and the results

were found negative. The reason for this could be contamination with other DNA

of different samples run on the same PCR or the contamination of the surface

area. Some factors might influence these results such as storage of samples

leading to DNA degradation and poor handling of PCR product. However, PCR is a

sensitive and specific method for identification of micro organisms whether

positive results were not obtained in the present study.

Keywords: Campylobacter jejuni, Bacillus cereus, Polymerase chain reaction

1

Table of Contents

1. INTRODUCTION...................................................................................................2

2. LITERATURE REVIEW..........................................................................................2

2.3. C. jejuni........................................................................................................2

2.4. B. cereus......................................................................................................2

3. MATERIALS AND METHODOLOGY.......................................................................2

3.1. Sample Collection........................................................................................2

3.2. Isolation and Culture conditions...................................................................2

3.3. Identification of isolated bacterial strains....................................................2

3.3.1. Conventional Cultural methods..............................................................2

3.3.2. Polymerase Chain Reaction (PCR) assay................................................2

3.3.2.1. DNA extraction....................................................................................2

3.3.2.2. Agarose Gel Electrophoresis...............................................................2

3.3.2.3. 16S rRNA gene amplification..............................................................2

3.3.2.4. Agarose gel electrophoresis................................................................2

3.3.2.5. Purification of PCR product.................................................................2

3.3.2.6. PCR cycle sequencing.........................................................................2

3.3.2.7. Sequencing.........................................................................................2

4. RESULTS.............................................................................................................2

4.1. Isolation and Identification...........................................................................2

4.2. DNA extraction.............................................................................................2

4.3. PCR assay.....................................................................................................2

4.4. Sequencing..................................................................................................2

5. DISCUSSION.......................................................................................................2

REFERENCES..........................................................................................................2

2

Table of Figures

Figure 1: CCDA selective agar plates showing grey coloured small and big colonies..................................................................................................................2

Figure 2: C. jejuni showing sodium hippurate hydrolysis exhibited by blue-purple overlayer in the right side tube..............................................................................2

Figure 3: B. cereus strains showing β-haemolysis on blood agar...........................2

Figure 4: Agarose gel electrophoresis U.V. image after DNA extraction of both bacterial strains. Channel 1 represents negative control. Channel 4 represents C. jejuni and channel 6 represents B. cereus. Channels 2, 3, 4 and 5 represents experiment carried out by another colleague........................................................2

Figure 5: Agarose gel electrophoresis U. V. image showing one DNA band obtained from purified PCR product of each bacterial strain. Channel 3 is showing DNA band for C. jejuni and channel 4 is showing DNA band for B. cereus. The channel 1 represents negative control and channels 2,5,6,7,8 and 9 represents the experiment carried out by another colleague. Channel 10 represents the ladder(1Kb size).....................................................................................................2

Table of Tables

Table 1: Colony characteristics and biochemical test results for colonies on both selective media......................................................................................................2

3

1. INTRODUCTION

Food borne illness caused as a result of consuming food contaminated with

pathogenic bacteria or their toxins is a major concern to public health problem.

Also it has economic consequences such as medical treatment, business loss,

lawsuits and investigation of the outbreaks. In order to overcome these, it is

important to have a rapid and effective detection and identification of food borne

pathogens in food industry, food safety agencies, public health bureaus and

bioterrorism prevention organizations. This will not only help in controlling and

investigating food borne pathogens but also in improving food safety in the food

industry.

Major food-borne pathogens include Bacillus cereus, Listeria

monocytogenes, Staphylococcus aureus, Clostridium botulinum, Clostridium

perfringens, Campylobacter jejuni, Vibrio parahaemolyticus, Yersinia

enterocolitica, Escherichia coli O157:H7 and Salmonella enterica serovar

Typhimurium (Kim et al, 2008; Oh et al, 2009). According to Oh et al, (2009) the

foremost techniques such as direct plating methods and biochemical tests are

currently employed to identify these bacterial pathogens. But these conventional

methods are time-consuming, labour-intensive and expensive because separate

cultivation of each target species is essential. Thus conventional methods have

been replaced by more rapid and cost effective molecular techniques such as

PCR and other hybridization tests (Fukushima et al, 2007).

Among the food borne pathogens Campylobacter species have considered

to be the most frequent pathogen among which C. jejuni is responsible for most

of the food borne infection worldwide (Churruca et al 2007). It has also been

reported that there has been an increase in the incidence of Campylobacter

infection during the last decade in Japan, North America and Europe according to

Bin Jasass and Park, (2009). Spore forming bacteria in food is also responsible for

food poisoning thereby raising major safety and issues and one such spore

forming bacterium is Bacillus cereus that has been recognised as a causative

agent of food poisoning for more than 40 years (Ghelardi et al., 2002) B. cereus

has thus become a major factor in severe food poisoning out breaks worldwide

(Ombui et al., 2008).

The main aim of the present study was to isolate Campylobacter jejuni and

Bacillus cereus from retail food samples such as poultry chicken and to identify

both by PCR and conventional methods.

4

2. LITERATURE REVIEW

With the objective of isolation of C. jejuni and B. cereus and their detection by

using PCR as well as by traditional methods, the review of literature is as follows:

2.1. Food borne pathogens

Bacterial pathogens are the major burden of food-borne illness as well as a major

public health impact (Altekruse et al., 1997). It was found that there are over two

hundred known microbes that are capable of causing illness when ingested. In

2005, the World Health Organization (WHO) reported that 1.8 million people had

died from diarrhoeal diseases due to consumption of contaminated food and

drinking water. Moreover, in the industrialised world during the last 20 years the

food borne diseases caused by bacteria, parasites, viruses and prions have

occasionally achieved ample media attention. The knowledge of intestinal illness

caused by food borne pathogens is very limited. It was showed that between 50

and 60% of causative agents of intestinal infectious diseases are not identified by

general diagnostic tools. Owing to the lack of diagnostic tools, gastroillness

caused by Bacillus cereus which is a toxin producing bacteria are under

estimated. It was recognized that before 1960 the major causes of

gastrointestinal disease were Salmonella spp, Shigella spp, Clostridium botulinum

and Staphylococcus aureus. Clostridium perfringens and Bacillus cereus were

added during the 1960s. A burst of additions was found in the 1980s and 19990s

consisting of Campylobacter, Yersinia, Listeria monocytogenes and new strains of

Escherichia coli, Cryptosporidia and Cyclospora (Newell et al., 2010).

Over the years there has been considerable increase in travel and trade and thus

the risk of continuous distribution of pathogens. Due to this, it has become

important to the food industry to have reliable and rapid tests for the

identification of presence or absence or even the degree of contamination of

pathogens (Malomy et al., 2003).

2.2. Detection of food borne pathogens

Detection and isolation of food borne pathogens are generally difficult because of

the large number of contaminating bacteria and due to the small number of

relevant pathogenic bacteria (Olsen, 2000). Traditional and standard

determination of food for the presence of bacteria depends on the enrichment

and isolation of presumptive colonies with appropriate diagnostic artificial media.

5

And this is followed by respective biochemical and serological identification tests.

The International Organisation for Standardization (ISO) has developed many

standards for the detection of major foodborne pathogens by traditional methods

for example, Salmonella, Listeria monocytogenes, thermotolerant

Campylobacter, Escherichia coli O157 and Staphylococci. Even though the

traditional methods of identification are reliable and effective, these methods

require several days and weeks to obtain results. Also those bacteria identified

based on the phenotypic properties might not always be expressed; and if they

are expressed they will be difficult to interpret and classify. Also the cells which

are viable but on the other hand nonculturable cannot be detected as in case of

some stressed Campylobacter spp (Malorny et al., 2003).

According to Olsen (2000), the ways of detecting pathogenic bacteria in food

have changed with the development of new techniques during the last 20-30

years. And they have been developed to speed up detection of pathogenic

bacteria and to increase the sensitivity of the detection. The DNA-based

detection methods have known for around 20 years. Introduction of the

polymerase chain reaction (PCR) has found to be increasingly used in research in

food microbiology (Olsen et al., 1995).The advantages of PCR-based detection

are specificity, sensitivity, rapidity, selectivity and potential for automation.

These particular properties have encouraged researchers to use PCR in the

laboratories (Mateo et al., 2005; Malorny et al., 2003).

2.3. C. jejuni

Campylobacters are microaerophilic, Gram negative, slender curved or spiral

rods, appearing vibroid and motile with single polar flagellum (Veron and

Chatelain, 1973). Among the Campylobacter species C. jejuni and C. coli are

commonly found worldwide, of which C. jejuni is responsible for 80-90% of

enteritis infections (Burnett et al., 2002). A common cause of bacterial

gastroenteritis in humans is caused as a result of Campylobacter spp. The

consumption and the poultry handling are considered to be the risk factors in

taking on campylobacteriosis. The poultry related meat preparations are

sustainable to mishandling during preparation by the consumer and

Campylobacter spp are constantly isolated; intermittently at high contamination

level (Uyttendaele et al., 2006).

According to Sallam, (2006) industrialized and developed countries have reported

high levels of Campylobacter isolation from retail chicken. And in many

6

industrialized countries the incidence of campylobacteriosis exceeds that of

salmonellosis (Debretsion et al., 2007). There were outbreaks in Japan associated

with consumption of raw or undercooked chicken meat due to the ingestion of

raw and undercooked chicken meat contaminated with Campylobacter spp.

Recent studies had shown that chicken meats and by products are the major

sources of C. jejuni and approximately 60% of retail poultry meats and by-

products in Japan were contaminated with C. jejuni (Bin Jasass and Park, 2009).

Moreover, there were cases of cross-contamination from poultry during

preparation. In order to prevent Campylobacter-related gastroenteritis in humans

it is essential to monitor Campylobacter contamination in chicken meat. But

chicken meat harbour only small numbers of Campylobacter cells. Therefore, in

order to detect Campylobacter spp in chicken-meat samples it is important to

have an enrichment procedure and one such standard enrichment method

consists of Campylobacter-specific broth supplemented with blood under

microaerobic conditions.

Campylobacters are fastidious, capable of hydrolysing very little range of sugars.

Also they possess few biochemical characteristics which can reliably be used to

distinguish between species. C. jejuni is differentiated from other Campylobacter

spp. by its ability to hydrolyse hippurate (Burnett et al., 2002). The detection of

Campylobacter spp. in food include conventional methods such as culturing in

selective media at 42º C under microaerobic conditions followed by biochemical

tests for the identification of isolates. However, the phenotypic identification of

Campylobacter spp is difficult to understand. Thus molecular amplification

methods like PCR allow a sensitive and specific method for the detection of

Campylobacter spp (Churruca et al., 2007). Many number of conventional PCR

assays are available for the identification and characterization of Campylobacter

spp from a range of sample types such as water, food products, stools and

cultures with the use of a variety of gene targets (Debretsion et al., 2007).

Oligonucleotide primers based on certain genes that are specific for C. jejuni also

have been developed recently (Bin Jasass and Park , 2009).

2.4. B. cereus

Bacillus cereus is a Gram positive, rod shaped motile bacterium. The presence of

endospores attributes its occurrence in natural environment as well as for the

high frequency of isolation from contaminated raw and processed food products

(Ghelardi et al., 2002). Because of the resistant spores of B. cereus raw materials

7

devoid of B. cereus are rarely obtained and thus it is capable of contaminating a

variety of food products like cooked chilled meals, pastries fish, meat, milk, liquid

egg, oils and fats (Ceuppens et al., 2010). Gastrointestinal and non-

gastrointestinal diseases are caused by B. cereus. Two types of food poisoning

syndromes are caused; emesis and diarrhoea. Diarrhoeal poisoning is as a result

of heat-labile enterotoxins produced during the vegetative growth of B. cereus

while emetic type is due to small, heat and acid-stable cyclic dodeca-

depsipeptide cereulide (Ehling-Schulz et al., 2004).

B. cereus is not a reportable disease, also the reporting procedures vary between

countries and therefore the incidence of B. cereus food poisoning is downgraded

(Ehling-Schulz et al., 2004). In Norway, during 1990 it was found that B. cereus

was the common microbe being isolated from foodborne illness and it was also

responsible for 14% of the outbreaks in Finland. Investigation reports of

foodborne outbreaks in the German Federal Armed Forces showed that B. cereus

was the frequently isolated pathogen in the retained food samples according to

Ehling-Schulz et al., (2004). And it was between 1985 and 2000, 42% of

outbreaks was reported due to B. cereus. The diarrhoeal type of food poisoning

was prevalent in Norway, Finland and Hungary and the emetic type was

predominant in the UK, Japan and the USA (Ehling-Schulz et al., 2004).

It is difficult to discriminate the genotypic and phenotypic features of closely

related species of B. cereus. Some of the specific features were targeted wit the

use of standard microbiological and biochemical methods which consists of API

tests. But these methods were limited to particular applications. Therefore

development of molecular tools such as sequencing of 16SrDNA, DNA-DNA

hybridization and PCR were used to identify various spore forming bacteria

(Postollec et al., 2010).

3. MATERIALS AND METHODOLOGY

3.1. Sample Collection

In the present study, two types of chicken samples including raw chicken (thigh

and breast) and processed chicken (thigh and breast) were collected from retail

food market. The samples were kept under refrigerated conditions at 40C until

isolation.

8

3.2. Isolation and Culture conditions

To isolate C. jejuni, 10g slices of each chicken sample (raw and processed) was

placed in a stomacher bag with 100ml Bolton broth (CM0983, Oxoid)

supplemented with bolton broth selective supplement (SR0183, Oxoid) and were

incubated at 370 C for 4h under microaerobic conditions (5% oxygen, 10% carbon

dioxide, and 85% nitrogen). After 4h, the samples were further incubated at 42oC

for 24h. Then 10gm each of these enriched samples including raw and

processesd chicken thigh and breast were homogenized for 30s in 90 ml of 0.1%

buffered peptone water (CM0509, Oxoid) using a stomacher and serial dilutions

ranging from 10-1-10-6 were prepared aseptically. Thereafter, 100 µl of dilutions

10-1-10-3 were plated onto duplicate plates of campylobacter blood-free agar base

(CM0739, Oxoid) supplemented with CCDA selective supplement (SR0155, Oxoid)

and 5% laked horse blood using spread plate method. The plates were then

incubated anaerobically at 37oC for 24h.

To isolate B. cereus, 10g of each chicken sample (raw and processed) was

homogenized in buffered peptone water (CM0509, Oxoid) for 30s using a

stomacher and serial dilutions (10-1-10-6) were prepared. Thereafter, 100 µl of 10-

1-10-3 dilutions of the samples were seeded onto duplicate plates of B. cereus

selective agar base (CM0617, Oxoid) supplemented with polymyxin B supplement

(SR0099, Oxoid) and the plates were incubated aerobically at 37oC for 24h.

The isolation procedure was repeated three times on both of the selective media

and the results of viable counts were taken as average of the three repeated

isolation. These isolated strains of C. jejuni and B. cereus were kept in brain heart

infusion broth and sub cultured every week until completion of the experiment.

3.3. Identification of isolated bacterial strains

3.3.1. Conventional Cultural methods

The isolated colonies of C. jejuni and B. cereus from selective media were

diagnosed for presumptive identification based on the colonial appearance and

by performing Gram staining; and biochemical tests such as catalase and oxidase

as described by Mackie and McCartney (1989). Sodium hippurate test was

performed to confirm the presence of C.jejuni due to its ability to hydrolyse

sodium (Roberts & Greenwood, 2002; Bayliset al., 2000). To perform sodium

hippurate test, the bacterial colonies from CCDA selective agar were taken and

subcultured on blood agar plates. After overnight incubation, the colonies from

9

blood agar plates were collected and shaken in 0.5 ml of 1% sodium hippurate

solution followed by 2 hr incubation at 370C in a water bath. Thereafter, at the

top of the hippurate solution in each tube, 0.2 ml of ninhydrin solution was added

and the tubes were incubated at 370C for 10 minutes to observe colour

development (Denis et al., 1999).

The bacterial strains from B. cereus selective agar plates were sub cultured onto

blood agar plates to detect β-haemolysis on blood agar. API 50CH test was

performed for the presumptive identification of B. cereus.

3.3.2. Polymerase Chain Reaction (PCR) assayThe isolated strains of C. jejuni and B. cereus identified by the conventional

methods were also subjected to perform PCR. The steps were performed as

follows:

3.3.2.1. DNA extraction

Chromosomal DNAs of both the isolated bacterial strains were extracted from

overnight BHI broth cultures as described by Ghelardi et al. (2002). In case of C.

jejuni, 1 ml of the grown BHI broth culture was taken into a pellet and centrifuged

at 8,000 rpm for 5 min and the supernatant was discarded. The cells were

resuspended in 500 µl of TES buffer (50mM Tris-HCl, pH 8.0, 10mM EDTA, 50 mM

NaCl) and 5µl lysozyme (20mg/ml) was added and the mixture was incubated at

37oC for 15-30 minutes, thereafter 5µl of Ribonuclease A (10mg/ml) and

Proteinase K (10mg/ml) were added one by one and incubated at 65oC for 20 min

respectively and vortexed. Then 50µl 20% SDS (sodium dodecyl sulphate)

following 600µl of phenol were added and the mixture was centrifuged at 8,000

rpm for 10 min to obtain DNA solution which was then resuspended into TE buffer

and stored at -20oC (Sanger and Coulson, 1975). On the other hand, for B. cereus,

the chromosomal DNA extraction was performed according to the instructions

given in QIAamp Mini Kit. 1ml of overnight BHI broth culture was centrifuged at

5,000 ×g for 10 min and supernatant was discarded. The cells were resuspended

in 180 µl of appropriate enzyme solution (20mg/m lysozyme; 20 mM Tris HCl,

pH8.0; 2mM EDTA; 1.2% Triton) and incubated at 37oC for 30 min. After that 20µl

of proteinase K and 200µl buffer AL and incubated at 56 oC for 30 min following

further 15min at 95oC leading to DNA degradation. This mixture was then

centrifuged for few seconds to obtain DNA.

10

3.3.2.2. Agarose Gel Electrophoresis

Agarose gel electrophoresis was performed to check the condition of the

chromosomal DNA and to get an approximate estimate of the DNA concentration

of both of the bacterial strains. 10µl of obtained chromosomal DNA extract of

both C. jejuni and B. cereus were run on 1% agarose gel after mixing each with

2µl of loading dye making the load upto 7 µl of DirectLoad Wide Range DNA

Marker. When the electrophoresis was complete, the gel was removed and

stained by soaking in ethidium bromide for 30 min at room temp after which gel

was placed on UV lightbox and photographed.

3.3.2.3. 16S rRNA gene amplification

Oligo primers including pA: 5’-AGA-GTT-TGA-TCC-TGG-CTC-AG-3’ (forward primer)

and pE: 5’-CCG-TCA-ATT-CCT-TTG-AGT-TT-3’ (reversed primer), based on

conserved regions of the 16S rRNA gene, were used to direct PCR amplification of

a 940 bp portion of this gene. PCR amplification was conducted in a 49µl reaction

mixture by using recombinant Taq DNA polymerase. Amplification of DNA was

performed in a DNA thermal cycler and after an initial denaturation at 950C for 5

minutes, 35 cycles of amplification were performed under the following

conditions: 940C for 1 minute, 550C for 1 minute, 720C for 1 minute followed by a

final extension at 720C for 5 minutes.

3.3.2.4. Agarose gel electrophoresis

The products of each reaction mixture of PCR were separated by subjecting 5 µl

aliquots to agarose gel electrophoresis (1% agarose) for 45 minutes at 100V

followed by 30 minutes staining in ethidium bromide solution with final

visualization and photography under UV light. Negative control sample was also

run for PCR amplification under the same conditions without adding the

chromosomal DNA template of bacterial strains.

3.3.2.5. Purification of PCR product

These PCR products of both bacterial strains including C. jujuni and B. cereus

obtained from amplification reactions were cleaned and purified for 30 minutes

by using a QIAquick PCR purification Kit (Qiagen) according to the manufacturer

instructions.

3.3.2.6. PCR cycle sequencing

The purified PCR products were then sequenced using oligo primer pD: 5’-GTA-

TTA-CCG-CGG-CTG-CTG-3’ to generate 550 bp of nucleotides. The sequencing

11

was done with the ABI Big Dye Terminator v 3.1 Cycle sequencing kit (Applied

Biosystems) by using 10 µl of the reaction mixture. Amplification of DNA was

performed in a DNA thermal cycler and after an initial denaturation for 2 minutes

at 950C, 35 cycles of amplification were performed under the following

conditions: for 15 seconds at 960C, 1 second at 400C followed by a final extension

of 4 minutes at 600C. Negative control was also run without adding PCR product.

These extension products were then precipitated using 1µl of 3M sodium acetate,

pH 4.6 and 50µl 100% ethanol.

3.3.2.7. Sequencing

The PCR product was sent to Oxford University for run on sequencing gel to know

the gene sequence. The obtained gene sequences were compared to the

nucleotide databases online using NCBI BLAST tool.

4. RESULTS

4.1. Isolation and Identification

Out of raw and processed chicken samples, only the raw chicken samples

including chicken thigh and chicken breast showed the presence of Gram-

negative rods on CCDA selective agar and Gram-positive rods on B. cereus

selective agar identified by Gram staining (Table 1). Two types of colonies

including small and big colonies showing grey colour on CCDA agar (Fig.1) were

observed. Gram staining results, viable counts obtained and biochemical test

results are shown in Table 1.

Table 1: Colony characteristics and biochemical test results for colonies on both selective media

Sample Plating

media

Colony

morphology

Gram

staining

Viable

count

(CFU/gm

)

Catalas

e

Oxidas

e

Chicken CCDA agar

Big, Grey

coloured with

metallic sheen,

smooth

Gram-

negative

rods

1.3 X103 + +

12

thigh Small, Grey

coloured ,

smooth

Gram-

negative

rods

1.2 X102 - +

Chicken

breast

CCDA agar Grey coloured

with metallic

sheen, smooth

Gram-

negative

rods

1.1 X102 + +

Chicken

thigh

B. cereus

selective

agar

Large, raised

and opaque

colonies with

irregular

margins

Gram-

positive

bacilli

1.8 X103 + +

Small, slightly

raised and

opaque with

regular margins

Gram-

positive rods 1.4 X102 _ +

Chicken

breast

B. cereus

selective

agar

Large, raised

and opaque

colonies with

irregular

margins

Gram-

positive

bacilli

1.2 X103 + +

The bacterial strains isolated on CCDA selective agar

showing catalase positive reaction hydrolysed sodium hippurate on testing (Fig.2)

by exhibiting bluish- purple overlayer in the tube which confirmed the presence

of C. jejuni whereas catalase negative strains showed no hydrolysis.

On the other hand, the bacterial strains isolated on B.

cereus selective agar showing catalase positive reaction exhibited β-haemolysis

on blood agar plates (Fig.2) whereas catalase negative strains showed no

haemolysis on blood agar. Thereafter, API test results performed on catalase

positive strains yielded 92.3% B. cereus from both of the raw chicken samples

(thigh and breast).

13

Figure 1: CCDA selective agar plates showing grey coloured small and big colonies

Figure 2: C. jejuni showing sodium hippurate hydrolysis exhibited by blue-purple overlayer in the right side tube

Figure 3: B. cereus strains showing β-haemolysis on blood agar

4.2. DNA extraction

The isolated C. jejuni and B. cereus strains were further subjected for

identification by PCR assay and both of the bacterial strains (C. jejuni and B.

cereus) exhibited a chunk of DNA at the top just below the wells (Fig. 4) after

agarose gel electrophoresis.

1 2 3 4 5 6 7 DNA Markers

14

1 Kb Ladder

Figure 4: Agarose gel electrophoresis U.V. image after DNA extraction of both bacterial strains. Channel 1 represents negative control. Channel 4 represents C. jejuni and channel 6 represents B. cereus. Channels 2, 3, 4 and 5 represents experiment carried out by another colleague.

4.3. PCR assay

After DNA amplification, the purified PCR product (DNA extract) of both C. jejuni

and B. cereus exhibited one DNA band for each bacterial strain when subjected to

agarose gel electrophoresis (Fig. 5).

1 2 3 4 5 6 78 9 10 1Kb Ladder DNA Markers

15

Figure 5: Agarose gel electrophoresis U. V. image showing one DNA band obtained from purified PCR product of each bacterial strain. Channel 3 is showing DNA band for C. jejuni and channel 4 is showing DNA band for B. cereus. The channel 1 represents negative control and channels 2,5,6,7,8 and 9 represents the experiment carried out by another colleague. Channel 10 represents the ladder(1Kb size)

From the above figure 5, it can be seen that the approximate estimated size of the DNA bands obtained from both C. jejuni and B. cereus was near to 700bp which was less than the expected 940bp size.

4.4. Sequencing

The gene sequences obtained from the purified PCR product and compared to the nucleotides databases using NCBI blast but they were not clearly seen and were overlapping unidentified nucleotides.

5. DISCUSSION

Campylobacter jejuni and Bacillus cereus have been associated with food borne

illness. Campylobacter jejuni cause human gastroenteritis and has received

increasing attention during the last two decades worldwide (Oh et al., 2008). And

Bacillus cereus has been recognized as a causative agent of food poisoning for

more than forty years which results in emetic and diarrhoeal syndromes

(Ghelardi et al., 2002).

The aim of this present study was to isolate, detect and identify

Campylobacter jejuni and Bacillus cereus with the help of sensitive, specific and

rapid polymerase chain reaction (PCR) as well as by conventional methods. Raw

and processed chicken samples, each containing thigh and breast was collected

from retail food market. The preliminary tests were performed by conventional

methods in which the results showed that only raw chicken samples were found

to show the growth of C. jejuni and B. cereus during third attempt with a CFU

ranging from 102 to 103 CFU/gm respectively. Thus all the raw chicken samples on

a third attempt of the same samples were found to be positive with the highest

C. jejuni count and B. cereus counts in chicken thigh compared to chicken breast

based on the culturing methods (Table 1). The present finding of C. jejuni was

also in agreement with those reported by Sallam (2007) in which C. jejuni was the

most prevalent species identified from chicken products such as chicken thigh,

breast, liver etc. The study also showed the occurrence of B. cereus and C. jejuni

in raw chicken samples as the spores of B. cereus would have been killed by heat

treatment. This occurrence of B. cereus in raw chicken samples is similar to the

finding of Eglezos et al. (2010). The study showed that only raw chicken samples

enhanced the growth of these bacteria while processed chicken samples were

16

devoid of these micro organisms. This is in contrast to the observation of

Guinebretiere et al. (2003) in which it was reported that bacterial spores were not

killed even after heat treatment.

As mentioned, only during the third attempt these bacteria were found to show

growth. The reason for this could be that refrigeration temperatures allowed the

growth of these microorganisms. The survival of C. jejuni under refrigeration

storage in this study was in agreement with the previous studies of El-Shibiny et

al., (2009) and B. cereus also found to survive in raw chicken at refrigeration

temperatures. Studies have shown that the spores of B. cereus are capable of

germinating and growing at refrigeration temperatures Valero et al., (2007). The

absence of these microorganisms in first two attempts also might be due to

erroneous procedure of isolation during serial dilutions, pipetting or during

plating.

From the results of biochemical testing, it can be seen that colony

characteristics, Gram-negative rods exhibited by Gram staining, catalase positive

reaction and hydrolysis of sodium hippurate confirmed the presumptive

identification of C. jejuni (Harvey, 1980 and Luechtefeld et al., 1982) on CCDA

selective agar plates. On the other hand, the presence of B. cereus was also

identified on the basis of catalase positive reaction, Gram staining showing Gram-

positive bacilli and API 50 CH profile further confirmed the presence of B. cereus

isolated from B. cereus selective agar. Thus conventional methods identified the

presence of both of the bacterial strains on selective media and further detection

was done with the use of PCR.

The PCR assay has been found to be a very sensitive and rapid method. Previous

studies have shown that rapid and effective detection of these food borne

pathogens can be achieved by PCR assays (Oh et al., 2008). The results of this

analysis by PCR amplification generated with the use of specific oligo primer sets

derived from the 16S rRNA, were found to be negative and the length of the DNA

bands obtained for both C. jejuni and B. cereus after DNA amplification was less

than the expected 940bp. Hence, a likely explanation for this could be

contamination which might have resulted from the DNA of other test samples or

due to contaminated surface area as PCR being so sensitive it is susceptible to

17

contamination. Also, repeated amplification of the same target sequence can

lead to accumulation of amplification products (Aslanzadeh, 2004). Thus the

sensitivity of PCR explains that even a low level of contamination with the target

DNA will result in positive signals (Fox et al., 2002).

However, it could be taken into consideration that PCR offers rapid and

effective identification of these food borne pathogens whether negative results

were obtained in the present study which might be due to poor handling of the

PCR product or long storage of the samples.

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