salmonella enteritidis salmonella typhimurium … of salmonella enteritidis and... · detection of...
Post on 19-Mar-2019
282 Views
Preview:
TRANSCRIPT
DETECTION OF SALMONELLA ENTERITIDIS AND SALMONELLA TYPHIMURIUM FROM WATER AND ANIMAL
SAMPLES IN KUBAH NATIONAL PARK, KUCHING, SARAWAK
Dellroy Donny
QR 101
Bachelor of Science with Honours S15 (Resource Biotechnology) D358
2012lOll
,.. \. \ I i·, ..
Pusat Khidmat MakJumat Akademik VNlVERSm MALAYSIA SAftAWAK
Detection of Salmonella enteritidis and Salmonella typhimurium From Water and Animal Samples in Kubah National Park, Kuching, Sarawak
P.KHIDMAT MAKLUMAT AKADEMIK
111111111 111111111 1000:235571
DELLROY DONNY 23374
The project is submitted in partial fulfilment of the requirement for the degree of Bachelor
of Science with Honours
(Resource Biotechnology)
Department of Molecular Biology
Faculty ofResource Science and Technology
Universiti Malaysia Sarawak
2012
...
l .' To .,
ACKNOWLEDMENTS
First, I would like to thank to Department of Molecular Biology, Universiti
Malaysia Sarawak for giving me the opportunity to fulfill my Final Year Project (FYP)
with the facilities provided. I really appreciate for all materials, equipment, instruments,
and other facilities provided which is very necessary for the completion of my project.
I would like to express my deepest gratitude and thanks to my supervisor, Dr.
Lesley Maurice Bilung and my co-supervisors, Professor Dr. Kasing Apun and Dr. Samuel
Lihan, for their advice, encouragement and guidance throughout my final year project. Not
to forget the postgraduate students in the Microbiology laboratory, Velneti Linang and
Christy Chan Sien Wei for the help and useful advices regarding the lab work.
Finally, I would also like to thank all my colleagues for their ideas, advices and
collaborations. I appreciate the valuable experience, knowledge and laboratory skills that I
gained during this project.
I 0 .'
DECLARATION
I declare that this thesis entitled 'Detection of Salmonella enteritidis and
Salmonella typhimurium from water and animal samples in Kubah National Park, Kuching,
Sarawak' submitted to the Faculty of Resource Science and Technology, University
Malaysia Sarawak (UNIMAS) is a presentation of my original research work and that is
has not been submitted anywhere except as cited in the references. This work was done
under the guidance of Dr. Lesley Maurice Bilung and this report work is submitted in the
partial fulfillment of the requirement for the degree of Bachelor of Science with Honours
in Resource Biotechnology.
Dellroy Donny
Faculty of Resource Science and Technology
Department of Molecular Biology
University Malaysia Sarawak
II
, ., .' " '" Pusat Khidmat Maklumat Akademik UNIVERSm MALAYSIA SARAWAK
TABLE OF CONTENTS Page
Acknowledgement I
Declaration II
Table of Contents III
List of Abbreviations v
List of Tables VI
List of Figures VII
Abstract VIII
Introduction I
1.1 Background
1.2 Research problems 3
1.3 Objectives 3
2.0 Literature review 4
2.1 Nomenclature / Taxonomy ofSalmonella 4
2.2 Salmonella infection outbreaks 5
2.3 Multiplex peR 6
2.4 Multiplex peR for detection ofSalmonella typhimurium 6
2.5 Multiplex peR for detection ofSalmonella enteritidis 7
3.0 Materials and methods 9
3.t Sample collection 9
3.2 Isolation and identification of presumptive Salmonella species 12
3.2.1 Enrichment ofbacterial samples 12
3.2.2 Isolation of Salmonella species on different type of selective agar 12
III
.' .'
3.2.3 Preparation of stock and working culture 13
3.2.4 Gram staining 13
3.4 Molecular analysis 14
3.4.1 DNA extraction 14
3.4.2 Multiplex peR 14
4.0 Results and discussion 16
4.1 Isolation and identification of presumptive Salmonella species 16
4.1.1 Isolation of Salmonella species on different type of selective agar 16
4.1.2 Gram staining 24
4.2 Molecular analysis 25
5.0 Conclusion and recommendation 30
References 31
Appendices 35
IV
LIST OF ABBREVIATIONS
PCR Polymerase Chain Reaction
JUC flagellar antigen gene
sefA fimbrial protein gene
g gram
Ilm micrometer
III microliter
IlM micromolar
mM milimolar
ml milliliter
U unit
°C degree Celsius
DNA deoxyribonucleic acid
% percent
LB Luria-Bertani
XLD Xylose-Lysine-Deoxycholate
MgCh magnesium chloride
dH20 distilled water
UV ultra violet
v
• • t I
LIST OF TABLES
Table Description Page
Table 2.1 Salmonella Nomenclature. 4
Table 3.1 List of samples positive for presumptive Salmonella spp. 11
Table 3.2 List ofwater and sediment samples positive for Salmonella spp. 12
Table 3.3 The specific peR amplification conditions of Salmonella spp. 15
Table 3.4 Oligonucleotide sequences of primers used for specific peR reaction. 15
Table 4.1 Observation of bacteria colonies grown on different selective agar. 17
Table 4.2 Appearance of organisms grown on different type of selective agar. 23
Table 4.3 Isolates which were chosen for multiplex peR. 25
VI
I •1 I
Figure
Figure 3.1
Figure 4.1
Figure 4.2
Figure 4.3
Figure 4.4
Figure 4.5
Figure 4.6
Figure 4.7
,
LIST OF FIGURES
Description Page
Maps showing the sampling sites. 10
Colonies of presumptive Salmonella spp. on XLD agar. 21
Colonies of presumptive Salmonella spp. on Salmonella 21
Chromogenic Agar Base.
Colonies of presumptive Salmonella spp. on Hektoen Enteric 21
Agar.
Gram-stain image of presumptive Salmonella spp. viewed under 24
light microscope
Amplicon obtained by multiplex PCR for detectingfliC and sefA 26
genes analyzed with 1.5% agarose gel electrophoresis.
Amplicon obtained by multiplex PCR for detectingfliC and sefA 26
genes analyzed with 1.5% agarose gel electrophoresis.
Amplicon obtained by multiplex PCR for detectingfliC and sefA 27
genes analyzed with 1.5% agarose gel electrophoresis.
VII
, .'
Detection of Salmonella enteritidis and Salmonella typhimurium from water and animal samples in Kubah National Park, Kuching, Sarawak
Dellroy Donny
Resource Biotechnology Programme Department of Molecular Biology
Faculty of Resource Science and Technology Universiti Malaysia Sarawak
ABSTRACT
Salmonella can cause zoonotic diseases which are transmitted between human and animals. Infection of Salmonella is called salmonellosis and most infected people may develop diarrhea, abdominal cramp and nau ea. The conventional method for detecting Salmonella serovars needed 4 to 7 days for a complete diagnosis. This study is carried out to detect the presence of S. enteritidis and S. typhimurium by using rapid method, the multiplex PCR technique. The samples were collected from anal, cloacal, feces and intestine of different type of animals including bat, squirrel, mongoose and bird in Matang Wildlife Centre and Kubah National Park, Kuching, by using swab method. Moreover, samples from water and sediment from Sungai Rayu, Matang are also taken. The samples were first inoculated into an enrichment media, Luria-Bertani broth and then cultured onto three types of selective agar; Xylose-lysine-Deoxycholate (XLD), Salmonella Chromogenic Agar Base and Hektoen Enteric (HE) agar, for isolation and identification of Salmonella spp. There are 83.3% of the total isolates which were identified as presumptive Salmonella spp. Next, the DNA extraction was carried out using cell boil method. Two primer pairs which target the sefA andjliC gene of S. enteritidis and S. typhimurium respectively, were used in the multiplex PCR. The results showed that all the isolates did not possess the targeted genes, which suggest the presence of other Salmonella serovars.
Key words: S. typhimurium, S. enteritidis, multiplex PCR, Matang Wildlife Centre, Kubah National Park
ABSTRAK
Salmonella boleh menyebabkan penyakit zooonotik yang tersebar di antara manusia dengan haiwan. Jangkitan daripada Salmonella dikenali sebagai salmonellosis dan menyebabkan cirit-birit, kekejangan abdomen dan rasa loya. Kaedah konvensional untuk mengenal pasti serotip Salmonella memerlukan 4 hingga 7 hari untuk mendapatkan diagnosis yang lengkap. Kajian ini dijalankan untuk mengesan kehadiran S. enteritidis dan S. lJphimurium dengan menggunakan kaedah multiplex PCR. Sampel-sampel diambil daripada dubur, kloaka, najis dan usus beberapa jenis haiwan yang berbeza iaitu kelawar, tupai, cerpelai dan burung di Matang Wildlife Centre and Kubah National Park, Kuching dengan menggunakan kaedah swab (mengesat). Selain itu, sampel daripada air dan mendapan dari Sungai Rayu, Matang juga diambil. Sampel-sampel tersebut diinokulasi dalam kaldu LB dan dikultur ke atas 3 jenis agar berpilih; iaitu XLD, Salmonella Chromogenic Agar Base dan HE agar untuk pengasingan dan identijikasi spesis Salmonella. Terdapat 83.3% isolasi yang dikenal pasti mempunyai kemungkinan sebagai spesis Salmonella. Pengekstrakan DNA dibuat dengan menggunakan kaedah pendidihan sel. Dua set primer yang mensasarkan gen sefA dan jliC, masing- masing daripada S. enteritidis dan S. typhimurium digunakan dalam multiplex PCR. Hasil eksperiment menunjukkan tiada isolasi yang memiliki gen yang disasarkan, menandakan kemungkinan kehadiran serotip Salmonella yang lain.
Kalil kunci: S. tyuhimurium, S. enteritidis, multiplex PCR, Matang Wildlife Centre, Kubah National Park
VIII
1.0 INTRODUCTION
1.1 Background
Bacteria of the genus Salmonella are gram-negative, rod-shaped, and non-spore-forming.
Salmonella species are also capable to grow either with the presence or absence of oxygen.
All members of the genus are motile with the diameters about 0.7 to 1.5 J,lm, 2 11m to 5 11m
in length and can grow between 7°C to 45°C. According to Hendriksen (2003), the
metabolic characteristics of Salmonella are hydrogen sulfide producing, urea negative,
oxidase negative, indole and Voges Proskauer (VP) negative, catalase positive, Simmon
citrate and methyl red positive, and also glucose fermentation with the production of acid
and usually gas (except for Salmonella typhi).
Salmonella can cause a widespread range of diseases called salmonellosis. Salmonella
enteritidis and Salmonella typhimurium have been reported as the major contribution to
human salmonellosis (Fries et al., 2003). Infection of Salmonella are zoonotic that may
occurs to human, birds, reptiles and can be found in cold and warm blooded animals, as
well as in the environment (Mirmomeni et al., 2009). Salmonellosis transferred via
infected food and water as well as through the fecal-oral route. Chen et al. (2010) stated
that the symptoms of salmonellosis are diarrhea, vomiting, mild fever, nausea, stomach
pain and headache. The sources of infection are usually from undercooked meat, poultry,
infected eggs, and unpasteurized milk products. Insects or birds may also cause Salmonella
contamination to foods. Studies of Salmonella from environmental samples indicate that
water is an important source of contamination, particularly irrigation water containing
manure, wildlife feces or sewage effluents (Castro-Rosas et al., 2011).
1
" ,fl'
According to Pui et al. (2011), salmonellosis continues to be the main public health
problem globally and gives negative influence on the economic as the cost of surveillance
investigation, treatment and prevention of illness increases. In the United States, it is
estimated that 1.2 million non-typhoidal Salmonella infections occur annually, resulting in
19 336 hospitalizations and 378 deaths, with the medical expenses and loss of productivity
cost between $0.5 and $2.3 billion dollars (Franklin et al., 2011). Research done by
Modarressi and Thong (2010) showed that in Malaysia, the most common non-typhoidal
Salmonella reported for the period of 2003-2005 by National Public Health Laboratory are
S. enteritidis (28.1 %), S. weltevreden (25.7%), s. corvallis (10.3%) and S. typhimurium
(6.7%). Moreover, in 2006, the incidence rate of typhoid and paratyphoid in Malaysia is
0.77 per 100,000 populations (Hamdan et al., 2008).
Established conventional methods used to detect Salmonella such as the laborious
bacteriological and serological identification needed at least 4 to 7 days for positive results
(Akiba et al., 2011; Pathmanathan et al., 2004). Therefore, alternative rapid methods are
required for diagnostic treatment. Currently, rapid method that utilize technologies such as
immunomagnetic separation, EIA- and ELISA-based assay incorporating fluorescent or
colorimetric detection and molecular techniques such as PCR-based assays and DNA
hybridization has been developed. PCR test is now widely applied as it can be used to
distinguish between bacterial strains.
Salehi et al. (2011) stated that in order to colonize a host cell, bacterial pathogen uses
adhesive appendages called fimbriae which bind glycoprotein or glycolipid receptors on
epithelial cells. Bacterial adherence is generally believed to be a prerequisite for infection
and have been associated with Salmonella pathogenicity. According to Mir et al. (2010),
the virulence of Salmonella depends on varies factors,encoded by genes which lead to
their invasiveness, colonization, intracellular survival and damage towards the host tissues.
2
I I.' " .
1.2 Research Problems
This study was intended to investigate the prevalence of S. enteritidis and S. typhimurium
in animals and water bodies at three sampling sites; Kubah National Park, Matang Wildlife
Centre and Sungai Rayu, Matang. The selected areas are places for recreational activities,
thus this research also concerned with the spreading of zoonotic disease with the risk of
transmitting the organism and disease to human (workers and visitors at the recreational
park). On the other hand, since wild birds and bats have the ability to fly freely to any
places, they can easily transmit the pathogenic bacteria to human.
1.3 Objectives
This study was undertaken with the following objectives:
1. To determine the occurrence of S. enteritidis and S. typhimurium using 3 types of
selective agar which are XLD, HE agar and Salmonella Chromogenic Agar Base,
from samples collected in Kubah National Park, Matang Wildlife Centre and
Sungai Rayu, Matang.
2. To screen and examine the pathogenic S. enteritidis and S. typhimurium from the
animal and water samples by using multiplex PCR technique.
3
I "'
2.0 LITERATURE REVIEW
2.1 Nomenclature / Taxonomy of Salmonella
The genus of Salmonella is a member of Enterobacteriaceae and consists of only two
species, Salmonella enterica and Salmonella bongori (Kim et ai., 2006). S. enterica
subspecies include enterica, salamae, arizonae, diarizonae, houtenae and indica. This
genus also further divided into approximately 2,500 serovars which distinguished by their
somatic (0) and flagellar (H) antigens.
According to Centres for Disease Control and Prevention (CDC) (2008),
Salmonella serotype is based on the immu_ne reactions with two surface structures; 0 and
H antigens. 0 antigen is a carbohydrate antigen and is the outermost component of
lipopolysaccharide. Meanwhile, H antigen is a protein antigen called flagellin which is
present in the flagella. Table 2.1 shows the nomenclature for Salmonella spp. while
appendix 6 shows their scientific classification.
Table 2.1 Salmonella Nomenclature (adopted from Su & Chiu, 2007)
Taxonomic Position (writing format) and nomenclature No. of Genus Species Subspecies Serotypes serotypes
(capitalized, (italic) (italic) (or serovars) in each italic) (capitalized, not species or
italic)* subspecies Salmonella enterica
bongori
enterica (or subspecies I)
salamae (or subspecies II) arizonae (or subspecies IlIa) diarizonae (or subspecies IIIb) houtenae (or subspecies IV) indica (or subspecies VI) subspecies V
Enteritidis, Paratyphi, Typhimurium 9,46:z:z39 43 :z29:6,7:I,v:l,5,7 21 :m,t:59:z36:13,22:z39
1504
502 95
333 72 13 22
*: Some selected serotypes (serovars) are listed as examples.
Subspecies I is present in both warm and cold blooded animals while other
subspecies are generally associated with cold-blooded animals.
4
Pusat Khidmat Maklumat Akademik UNlVERSm MALAYSIA SARAWAK
Most serotypes belong to the species S. enterica ssp. enterica. Some of the
serotypes belonging to this subspecies are described and named by the location of an
outbreak (e.g. S. enterica subspecies enterica serotype Montevideo, referred to as S.
Montevideo), while others are identified by their antigenic formula (Fries et al., 2003).
i.e subspecies 0 antigens: Phase 1 H antigen(s): Phase 2 H antigen(s)
e.g I 4,5,12:i:2
The official name for an isolate with the antigenic structure 4,5, 12:i:2 is Salmonella
enterica subspecies enterica serotype Typhimurium. However, this isolate is normally
called Salmonella Typhimurium (CDC, 2008).
2.2 Salmonella infection outbreaks
According to the Centres for Disease Control and Prevention (CDC) (2011), a total of 241
individuals were infected with the outbreaks strain of S. typhimurium since 1 st April 2009
until 18th July 2011 in United States. The infections are associated with African dwarf
flogs and water from their habitat. Besides that, the outbreak strain of S. enteritidis has also
been reported which infected 42 persons around 20th August 2011 until 27th September
2011. The infections are linked to Turkish pine nuts (CDC, 2011). CDC also reported that
a total of 19 persons range in age from 1 year to 79 years old, were infected with the
outbreak of a multi-drug resistant strain of S. typhimurium from 7 states in United States,
which began on or after October 8, 2011 until December 2011. The illness was related to
the consumption of ground beef.
5
Research done by Anita et al. (2009) reported that the typhoid outbreak in Sungai Congkak
Recreational Park, Selangor was resulted from contaminated river water due to the poor
sanitation. The river water was contaminated with sewage disposal from two public toilets
which effluent grew Salmonella spp. The outbreaks of typhoid fever in Malaysia are found
to be sporadic which confined to the areas with inadequate safe water supply and
sanitation, food handling and personal hygiene practices (Anita et al., 2009).
2.3 Multiplex PCR
Polymerase chain reaction (PCR) is a powerful tool in in vitro amplification of specific
DNA sequence which will generate minions of target sequence copies (Amini et al., 20 10).
Multiplex PCR is a technique which able to amplify multiple targets within a single PCR
mixture and produce outcomes of varies sizes. This technique contributes to the need for
rapid species identification and detection thus will save time and cost of sample processing
(Salem et al., 2010). In order for the PCR to work properly, the reaction conditions
including the type and concentration of enzyme, reaction buffer content, time and
temperature of the annealing/extension process must be optimized (Liang et al., 2011).
2.4 Multiplex PCR for detection of Salmonella typhimurium
Research has been done by Lim et al. (2003) on selective detection of S. typhimurium by
using multiplex PCR, which targeted on abequose synthase (rjbJ) gene, flagellar H: i (fIiC)
antigen gene and flagellar H: 1,2 (fIjB) antigen gene. In this study, DNAs from S.
typhimunum, 15 other Salmonella serovars, and 8 non-Salmonella enteric pathogens were
used for evaluation of the targeted genes. Multiplex PCR proved to be capable of
6
specifically identifying S. typhimurium and discriminate it from other Salmonella serovars
and also non-Salmonella enteric pathogens. The results of this experiment showed that
none of the other Salmonella serovars and non-Salmonella enteric pathogens gave positive
results for all of the three amplified products which derived from the rib}, fiiC, and fijB
genes, at the same time.
In addition, Kim et al. (2006) has carried out a research for identifying the common
clinical serotype of Salmonella enterica subspecies enterica by using multiplex PCR-based
method. Some of the serotypes gave unique amplification patterns when compared to each
other.
2.S Multiplex peR for detection of Salmonella enteritidis
Salehi et al. (2011) stated that bacterial pathogen uses adhesive appendages called fimbriae
which bind glycoprotein or glycolipid receptors on epithelial cells in order to colonize a
host cell. They are one of the significant characteristic which involves in the survival and
persistence in the host. Thus a research has been done to detect the sefl4, sefl 7 and sef21
fimbrial virulence genes of S. enteritidis by using multiplex PCR. The sefl4 fimbriae were
shown to be the T-cell immunogen which allows the adherence to mouse epithelial cells.
Moreover, sefl7 fimbriae are a thin aggregative fimbriae encoded by the agf operon.
Meanwhile, sef21 are nearly similar to the type 1 fimbrin of S. enteritidis and consists of
21 kDa fimbrin monomers. The samples of diarrheic feces were collected from 30 hens
and 30 others from livestock in different regions in Iran. The results showed that 45
samples which were positive for S. enteritidis presented identical bands which indicated
sefl4, sef17 and sef21 (Salehi et al., 2011).
7
Moreover, Yasmin et ai. (2007) performed a study on the simultaneous multiplex peR for
detection of E. coli, L. monocytogenes and S. enteritidis in shrimp samples which targeted
on their specific DNA sequence. Furthermore, multiplex peR carried out on the
commercially imported shrimp samples showed that none of them contain any of the three
pathogens. This outcome suggests that the multiplex peR is a reliable and beneficial for
rapid detection of bacterial pathogens thus will save time and increase ability to assure
food safety (Yasmin et ai., 2007).
8
3.0 MATERIALS AND METHODS
3.1 Sample coUection
Bat, squirrel, treeshrew, mongoose, bird, water and sediment samples were collected from
Kubah National Park, Matang Wildlife Centre and Sungai Rayu, Matang on 3 June 2011
by a team of UNIMAS postgraduate students. Kubah National Park is located about 20 km
west of Kuching and covers an area of 2, 230 hectares, and comprises of heavily forested
slopes and sandstone ridges. The park is consisting mostly of palms vegetation and is home
to a variety of wildlife. Meanwhile, Matang Wildlife Centre is situated at the western
comer of the Kubah National Park and covers around 180 hectares of lowland forest. This
centre is Sarawak's main centre of rehabilitation for endangered wildlife. Sungai Rayu is
located nearby the Kubah National Park. The land is not cultivated and most of the natural
vegetation is still intact. The landscape is mostly covered with rain-fed croplands. The map
showing the location of each sampling sites is shown in Figure 3.1.
9
Figure 3.1 Maps showing the sampling sites at Kubah National Park (A), Matang Wildlife Centre (8) and
Sungai Rayu (C) (Source: Goog\e Earth, 2012).
A total of 67 samples which consist of 52 anal swabs, 9 cloacal swabs, 5 fresh feces
and I small intestine samples were obtained from different species of bat, squirrel,
mongoose, and bird collected from Matang Wildlife Centre and Kubah National (Table
3.l). The samples were enriched into 2 ml Luria-Bertani (LB) broth at 37°C for 18-24
hours. The samples were subcultured on Xylose-Lysine-Deoxycholate (XLD) agar for the
isolation of Salmonella spp. Sixteen samples of anal swabs, three samples of cloacal and
two samples of feces were positive for the presence of presumptive Salmonella spp.
Collections of water and sediment samples were conducted at Sungai Rayu,
Matang. During the first sampling, four samples of water _and sediment were coUected and
another three samples of water and sediment were collected from the second sampling. A
10
11.
12.
13.
14.
15.
16.
series of dilution was conducted for the sediment samples. All the samples were
subcultured on XLD agar for the isolation of Salmonella spp. Three samples of sediment
were positive for the presence of Salmonella spp. while there was none from water
samples.
Table 3.1 List of samples positive for presumptive Salmonella spp.
Sample FieldAnimal Common Name Scientific name
ty~e number Short-nosed fruit
1. Bat TK172776 Cynopterus brachyotis Anal bat
Short-nosed fruit 2. Bat TK172777 Cynopterus brachyotis
bat
3. Bat TK172779 Papillose wolly bat Kerivoula papil/osa
4. Treeshrew TK172784 Large treeshrew Tupaia tana
5. Bat TK172786 Fawn roundleafbat Hipposideros cervinus
Spotted-winged6. Bat TK172794 Balionycteris maculata
fruit bat
7. Bat TK172797 Dusky fruit bat Penthetor lucasii
Short-nosed fruit 8. Bat TK172799 Cynopyterus brachyotis bat
Spotted-wingedBat TK172801 Balionycteris maculata
fruit bat Short-nosed fruit
Bat TK172802 Cynopyterus brachyotis bat
Spotted-wingedBat TK172809 Balionycteris maculata
fruit bat
Bat TK172815 Dusky fruit bat Penthetor lucasii
Spotted-wingedBat TK172824 Balionycteris maculata
fruit bat Short-nosed fruit
Bat Rl Cynopterus brachyotis . bat
Bat R2 Fawn roundleafbat Hipposideros cervinus
Short-tailedMongoose Ml Herpestes brachyurus
Mongoose
11
Table 3.1 List of samples positive for presumptive Salmonella spp.
Yellow rumped 17. Bird MTOOI Prionochilus xanthopygius
flowerpecker Red-tailed tailor
18. Cloacal Bird KN4 Orthotomus sericeus bird
Yellow bellied 19. Bird KNP7 A lophoixus phaeocephalus
bulbul TK172776 Short-nosed fruit
20. Bat Cynopterus brachyotis (F) bat
Feces TKl72786
21. Bat Fawn roundleafbat Hipposideros cervinus (Fl
Table 3.2 List of water and sediment samples positive for Salmonella spp.
Sampling no. Sample code Presumptive Salmonella spp. CSl-S 1
1st sampling UCSI-SI
2nd sampling UCS2-W
3.2 Isolation and identification of presumptive Salmonella species
3.2.1 Enrichment of bacterial samples
Enrichment of all bacterial samples was done using Luria Bertani broth (Laboratorios
Conda, Spain). Five hundred microliter of the sample was inoculated into Bijoux bottle
containing 3 ml of LB broth and incubated (SHEL LAB, USA) at 37°C, overnight.
3.2.2 Isolation of Salmonella species on different type of selective agar
A loop full of sample was streaked onto XLD agar, Hektoen Enteric Agar, and Salmonella
for overnight. The bacterial growth on the plates were observed and recorded.
12
3.2.3 Preparation of stock and working culture
Colonies of presumptive Salmonella spp. from XLD agar were picked and cultured onto
nutrient agar (NA) (Bendosen Laboratory Chemical, Norway) slant. The cultures were
prepared in duplicate for stock and working cultures.
3.2.4 Gram staining
Gram staining was conducted according to the protocol designed Rollins and Joseph
(2000). A loopful of distilled water was placed on the slide. Then, a loop of colony sample
was transferred to the water drop and emulsified. The film was allowed to dry then passed
briefly through the Bunsen flame. Next, the slide was covered with a few drops of crystal
violet staining solution for one minute and washed briefly with tap water. The slide was
then treated with a few drops of Gram's Iodine and allowed to act for one minute.
Subsequently, the slide was washed by using tap water and then decolorized in absolute
ethyl alcohol. Finally, the smear was treated with a few drops of safranin solution and
allowed to counterstain for 30 seconds, then washed off with tap water and dried blotting
paper. The characteristics of the bacteria then were observed under light microscope.
13
3.3 Molecular analysis
3.3.1 DNA extraction
The bacterial DNA extraction was conducted according to the boiling extraction method as
described by Bilung et al. (2005) with minor modification. Salmonella isolates was
cultured in 3 ml LB broth for overnight at 37°C with agitation at 145 rpm (New Brunswick
Scientific Innova 4000). One milliliter of the sample transferred to a 1.5 ml
microcentrifuge tube. The cell suspension was centrifuged (Hettich Zentrifugen, Germany)
at 10 000 rpm for 5 min. The supernatant was discarded carefully. The pellet was then
resuspended in 300 III of distilled water by vortexing (Labnet International, USA). The
microcentrifuge tube was incubated for 20 min at 100°C and immediately chilled on ice for
20 minutes. Next, the tube was centrifuged for 5 min at 13 400 rpm. The supernatant was
carefully transferred to a new micro centrifuge tube and used as the template DNA in the
peR.
3.3.1 Multiplex peR
Multiplex PCR for detection of Salmonella enteritidis and Salmonella typhimurium was
perfonned according to Jamshidi et al. (2009), with some modifications. The PCR was
carried out in a reaction volume of 25 III containing 2.5 III lOx reaction buffer (Invitrogen,
Brazil), 0.5 III Taq Polymerase (2.5 U) (Fermentas, International Inc., Canada), 0.5 IlM of
each primer (10 mM), 1.25 III dNTPs (10 mM) (RBC Bioscience Corp., Taiwan), 1.5 III
MgCh (50 mM) (Invitrogen, Brazil), and 2 III genomic DNA. The samples were subjected
to 3S cycles of amplification using Eppendoft Mastercyc1e® Personal. Table 3.3 and Table
4 shows the specific PCR amplification conditions of Salmonella spp. and the
~ucleotide sequences ofprimers used, respectively.
14
top related