Faculty of Resource Science and Technology
Isolation and Characterization of Shigella spp. from Water in Aquaculture Farm
Stephanie Lenja anak Sait (50973)
Bachelor of Science with Honours (Resource Biotechnology)
2018
Isolation and Characterization of Shigella spp. from Water in Aquaculture Farm.
Stephanie Lenja anak Sait (50973)
A thesis submitted in partial fulfilment of the requirement for the degree or Bachelor of Science with Honour (Resource Biotechnology)
Supervisor: P.rof. Kasing Apun Co-supervisor: Dr. Samuel Lihan
Resources Biotechnology Programme
Faculty of Resource Science and Technology Universiti Malaysia Sarawak
9June2018
DECLARATION
I hereby declare that no portion of this dissertation has been submitted in support of and application for another degree qualification of this or any other university or institution of higher learning.
UNIVERSITI MALAYSIA SARA WAK
Grade: ---
Final Year Project Report Masters PhD
Please tick(/) (V)
( ) ( ) DECLARATION OF ORIGINAL WORK
This declaration is made on the .}~~\~ day of
~~f
year -:-1.9.~ .Student's Declaration: I
joFkzie Leya at $i+ 450473on A@1ff e} esoycet Sci ece «ad Teck«oggy _ (PLEASE INDICATE NAME, MATRIC NO. AND FACULTY) hereby declare that the work entitled,
lsolahow and Cw@clevis@ion o# Ci qetfa pg; hyes@i@y ]Agua@ye {«rma is my original work. I have not copied from any other students' work or from any other sources with the exception where due reference or acknowledgement is made explicitly in the text, nor has any part of the work been written for me by another person.
\ fut )61& f}erkoi@Leuy ot(it (sot?3) Date submitted Name of the student (Matric No.)
Supervisor's Declaration:
I, Pi--u-f .
Pr
l<-a~·1n~ AP4n (SUPERVISOR'S NAME) herebycertify that the work entitled {ClrlA
to]aow ox.d Chara<tevit@hon 5{Ski9ei) pr iWatee Accaftr@ 4 (TITLE) was prepared by the aforementioned or above mentioned student, and was submitted to the
"FACULTY" as a partial/full fulfilment for the conferment of
llci.r~~,~r of (ge11r$, l-'vm ~01-101111 )(PLEASE INDICATE THE DEGREE TITLE), and the afor~~~'iiti6~ed1 ~~it't6~he best of my knowledge, is the said student's work Received for examination by:
r ro-\ · 0
rosinq Q
Y" Date: \ II(,
I "\N\f((Name of the supervisor)
II
(Please tick (V)):
D CONFIDENTIAL (Contains confidential information under the Official Secret Act 1972)*
D RESTRICTED (Contains restricted information as specified by the organisation where research was done)*
(/OPEN ACCESS
Validation of Project/Thesis
I hereby duly affirmed with free consent and willingness declared that this said Project/Thesis shall be placed officially in the Centre for Academic Information Services with the abide interest and rights as follows:
D This Project/Thesis is the sole legal property ofUniversiti Malaysia Sarawak (UNIMAS).
D The Centre for Academic Information Services has the lawful right to make copies of the Project/Thesis for academic and research purposes only and not for other purposes
D . • The Centre for Academic Information Services has the lawful right to digitize the content to be uploaded into Local Content Database.
D The Centre for Academic Information Services has the lawful right to make copies of the Project/Thesis if required for use by other parties for academic purposes or by other Higher Leaming Institutes.
D No dispute or any claim shall arise from the student himself/ herself neither a third party on this Project/Thesis once it becomes the sole property of UNIMAS.
D This Project/Thesis or any material, data and information related to it shall not be
distributed, published or disclosed to any party by the student himself/herself without first obtaining approval from UNIMAS.
Student's signature
An
Supervisor's signature
Daer
~G l ove
Current Address:
LOI 210 Loo@ &U /J@pan Vt&ayy Svj Anwar, yaw«
Notes:* If the Project/Thesis is CONFIDENTIAL or RESTRICTED, please attach together as annexure a letter from the organisation with the date of restriction indicated, and the reasons for the confidentiality and restriction. [The instrument was prepared by The Centre for Academic Information Services]
II
ACKNOWLEDGEMENT
I would like to take this opportunity to express my heartfelt gratitude and gratefulness to every person that given me a helping hand throughout the process of preparing this report.
First and foremost, I would like to acknowledge my supervisor of this study, Prof.
Kasing Apun for valuable advices and constant patience throughout the process of this final year project. Her constant encouragement has always been a moral support for me.
My sincere thanks to my co-supervisor Dr. Samuel Lihan for his concern throughout the project work.
Besides that, I would like to express my gratitude to our lab technician of Faculty Resource Science and Technology of Universiti Malaysia Sarawak especially Mr. Azis Bin Ajim, for sacrificing his time by providing material and equipment during conducting this study. Special acknowledgement toward all senior lab mates and loyal advisor, Jennifer Jalan, Aizuddin Bin Shahbudin, Ahmad Syatir, Victoria Ulok, Rosdi Anak Kira and Nurul Asyiqin Binti Jamil whom have been assisting me in searching information and conducting experiments as well as giving me encouragement to accomplish the project.
I also would to express my cordial thanks to my group final year project member, Nurul Nadia Binti Zayadi, Norsyazira Binti Kadri, Scholastica Ramih Anak Buya, and Laurie Anak Ali whom always be there· and been source of inspiration throughout the process of final year project.
Finally, I wish to extend a warm thanks to everybody involved directly and indirectly with my work. The other credits of my achievement goes to my family who always there for me in my difficulties. and their faith in me that had always helped me to proceed further.
IV
Isolation and Characterization of Shigella spp. from Water in Aquaculture Farm.
Stephanie Lenja Anak Sait
Resources Biotechnology Programme Faculty of Science and Technology
Universiti Malaysia Sarawak
Shigellosis, the clinical presentation for Shigella spp. infection is characterized by abdominal cramps, fever, and diarrhea included four groups S.sonnei, S.boydii, S.dysentery and S.flexneri, Therefore, Shigella are responsible pathogen for majority of bacillary dysentery cases. However, their presences in aquaculture sector had become a concern, especially affect towards to human health. Besides, treatment of shigellosis with appropriate antimicrobial agents is able to shorten duration of illness the consequences are they able to resists against antimicrobial agents are increasing. The general aim for this study was to isolate and identification the presences of Shigella spp. in both traditional and commercial aquaculture farm. In order to achieve this, water sampling collection from I traditional pond and 2 aquaculture pond, isolated by XLD agar, differentiated by several biochemical tests and molecular analysis (polymerase chain reaction). Amplification of Shiga toxin (stx gene) was done to confirm the presences of pathogenic Shigella spp. in both type farms. Three cultures samples from each aquaculture farm were assessed with some appropriate antibiotics such as ampicillin, tetracycline, sulphonamides, erythromycin and sulfamethoxazole.
Overall, the bacterial strain are able to resist the antibiotics.
Key words: Shigella spp., Aquaculture farm, biochemical tests, Polymerase Chain Reaction (PCR), stx gene, Antibiotics
Abstrak
Shigellosis, merupakan proses jangkitan untuk spesies Shigella yang mengakibatkan kekejangan abdomen, demam, dan cirit-birit. Spesies Shigella. dibahagikan kepada empat kumpulan termasuk spesies sonnei, spesies boydii, spesies dysentery dan spesies flexneri, dan ini menyebabkan terhasilnya Shigella kerana patogen yang bertanggungjawab dalam majoriti kes disentri bacillari.
Tetapi, kehadiran Shigella dalam sektor akuakultur telah menjadi kebimbangan, terutamany a dalam menjejaskan kesihatan manusia. Penggunaan antibiotik yang sesuai telah menyingkatkan tempoh penyakit tetapi kesan daya ketahanan terhadap antibiotik yang biasa digunakan semakin meningkat.
Matlamat utama dalam kajian ini adalah untuk mengasingkan dan mengenalpasti spesies Shigella di kedua-dua ladang akuakultur tradisional dan komersil. Bagi mencapai ini, sampel air dari I kolam tradisional dan 2 kolam akuakultur, diasingkan dengan agar XLD, dibezakan dengan beberapa kajian biokimia dan analisis molekul. Penyah- toksin Shiga (stx gen) dilakukan untuk mengesahkan kehadiran patogenik Shigella spp. dalam kedua-dua jenis kolam. Bakteria yang mengandungi toksin Shiga diuji kaji dengan ampicillin, sulfonamid, sulfametoksazol,erythromycin, dan tetracycline. Kesimpulannya, bakteria daripada ketiga-tiga kolam ini mampu mengatasi kesan- kesan antibiotik tersebut.
Kata kunci: spesies Shigella, Jadang aguakultur, gen stx, Antibiotik
V
Table of Contents
Declaration I-III
Acknowledgements IV
Abstract V
Abstrak V
Table of Content VI
List of Abbreviations VII
List of Tables VIII
List of Figures IX
1.0 INTRODUCTION 1-2
2.0 LITERATURE REVIEW 3
2.1 Aquaculture Farm 3
2.1.1 Microbes that developed antibiotic resistances in aquaculture 4 farm
2.2 Antibiotic and Antibiotic resistant-mechanisms 4-5
2.3 Disc Diffusion Method 5
2.4 Enteric Bacteria 6
2.5 Shigella spp. 6-7
2.5. l Shigella spp. in water 7
3.0 MATERIALS AND METHODS 8
3.1 Sample Collection 8
3.2 Enrichment of Water Samples 8-9
3.3 Isolation of Shigella spp. 9
3.4. Identification of Shigella spp. 9
3.4.1 Gram Staining 9
3.5 Biochemical Characterisation 10
3.5.1 Oxidase Test 10
3. 5 .2 Catalase Test 10
3.5.3 Citrate Test 10
3.5.4 Kligler Iron Agar Test 10
3.6 Molecular Characterisation 11
3.6.1 Genomic DNA Extraction · 11
3.6.2 Polymerase Chain Reaction 11-12
3. 7 Antibiotic Susceptibility Tests 13
4.0 RESULTS 14-1 7
5.0 DISCUSSION 18-21
6.0 CONCLUSION 22
7.0 REFERENCES 23-25
8.0 APPENDICES 26-32
VI
List of Abbreviations
Shigella spp. Shigella species it
CLSI Clinical and Laboratory Standards Institute
ST Shiga toxin
T3SS Type 3 Secretion System
ML Millimetre
RPM Revolutions per minute
dlH; O Distilled water
µI Microliter
mM millimole
MgCl; Magnesium Chloride
dNTPs deoxyribonucleic phosphate
TBE Tris-Borate-EDT A
PCR Polymerase Chain Reaction
rRNA Ribosomal Ribonucleic acid
EtBr Ethidium Bromide
w/v Weight per volume
UV Ultra violet
MHA Mueller- Hinton Agar
TSA Tryptone Soy Agar
CFU Colony Forming Unit
H»O, hydrogen peroxide
KIA Kligler Iron Agar
VII
List of Tables
Table Title Page No.
3.6.2.1 The stx forward and reverse primers sequences 11
3.6.2.2 The master mixtures for stx amplification 12
3.6.2.3 The PCR Programme for stx gene 12
4.1 Parameters of each aquaculture farm 14
4.2 Colony Forming Unit(cfu) 14
4.3 The antibiotics assessment against isolates 17
VIII
List of Figures
Figure Title Page No.
l
3.1 Four selected points within 1 pond 8
4.1 The bacteria structure under compound microscope 15 4.2 Agarose gel electrophoresis of extracted Shigella spp. 16
genomic DNA
4.3 Results of isolates tested with antibiotics 17
LX
1.0 INTRODUCTION
Aquaculture farming is an important sector in the agriculture industry and expected to grow further to meet the future demand. The rapid growth and high production of carnivorous species such as salmon, shrimp and catfish in which leads to globalizing trend especially in food agriculture.
The prevalent mechanisms applied in the aquaculture farm was the used of antibiotic. The usage of antibiotic in aquaculture farm mainly chemical substances have the capacity to kill or inhibit the growth of the microorganisms. Antimicrobial agents are widely used as treatment, therapeutic, prophylactic in fish farming (Aly & Albutti., 2014).
The antibiotic are usually used to increase the growth and as feed efficiency in animals and some of it are frequently used in both veterinary and human medicine (Kathleen et al., 2016).
In aquaculture, antimicrobials are regularly added to the feed, which is then placed in the water where the fishes are kept but in some cases, antimicrobials may be added directly to the water and based on Romero et al. (2012) to prevent the infection disease to spread over the aquaculture farm and with limited vaccine, it has tum to increase usage of the antimicrobial agents or antibiotic such as the prophylactic procedure.
However, the extensive establishment of aquaculture farm has led to extreme usage of antimicrobial which leads to the raise of concern in the human and environment health.
The heavy used of the antibiotics and synthetic antimicrobial agents contribute to the selective pressure (Suzuki et al., 2017). It shows that the antibiotic supposedly inhibit and kill the growth of microorganisms but it turns into other side which the bacteria living in aquaculture had resist toward the antibiotic. The over-use of antibiotic toward aquaculture microbes make them resistant to antibiotic and sanitary barrier used in terrestrial food
animal production are difficult to establish in aquaculture. This condition with combination of high population density, water quality, or both, may lead to increase of bacterial infection thus contribute to the increase of antimicrobials agent usage, thereby increasing the selective pressure on bacteria in aquatic environment (Heuer et al. 2009).
This study also concern on the. microorganisms Shigella spp. presence in the aquaculture farm. Shigella spp. can be transmitted through fecal-oral route where it affects the human colon, with an infectious dose of only 10-100 organisms (Levine et al., 2013).
In addition, virulence factor of Shigella spp. determines their specific mechanisms compare to other genera of Enteric bacteria. A greater understanding of antibiotic resistance in aquaculture farm is required as water is an important source for human activity hence could be a transport medium of Shigella spp. The antibiotics may not able to resist the Shiga toxin as the Shigella spp. strain may susceptible to antibiotics due to overuse-antibiotic.
The objectives of this project are:
I. To determine the presence of Shigella spp. in water of both traditional and commercial aquaculture farm.
2. To compare the antibiotic resistance of Shigella spp. isolated from water in
commercial and traditional aquaculture farm.
3. To distinguish the antibiotic resistant of stx genes-producing Shiga Toxin from water in commercial and traditional aquaculture farm.
2.0 LITERA TURE REVIEW
2.1 Aquaculture Farm
Aquaculture farm were known since back days which it focused on the farming of aquatic livings such as fish, crustaceans, mollucscs and microalgae. According to Bostock et al. (2010), the aquaculture had contributed 43 per cent of aquatic animal food for human consumption in 2007 and it expected to grow further to meet the future demand. The aquaculture sector has include several factors which made it became globalizing and trends in food industries. For instance, the output, value and the regional overview. The certain places has been driven by factors such as pre-existing aquaculture practices, population and economic growth, relaxed regulatory framework and expanding export opportunities (Bostock et al., 2010).
Besides, the growth rates of aquaculture and the species produced from freshwater also affect the high demand of aquaculture systems. Every aquaculture farm has it different systems and environment typically freshwater ponds and tanks, freshwater cages, coastal ponds and tanks, coastal cage farms, and marine molluscs and aquatic plants. In addition to their use in human medicine, antimicrobials are also used in food animals and aquaculture, and their use can be categorize as therapeutic, prophylactic or metaphylactic. Based on Romero et al.(2012), therapeutic use corresponds to the treatment of established infections, for metaphylaxis is a term used for group-medication procedures that aim to treat sick animals while also medicating others in the group to prevent disease and prophylaxis means the preventative use of antimicrobials in either individuals or groups to prevent the development of infections. Hence, the promotion of antibiotic towards aquaculture has being one of significant in aqua-farming but in certain limits and levels.
5
2.1.1 Microbes that developed antibiotic resistance in aquaculture farm
Large number of bacteria from highly diverse bacterial species could be isolated from aquaculture pond and it environment. The presences of highly diverse bacterial provide high level nutrients in the water (Kathleen et al., 2013). Based on previous study by Apun et al. ( 1999), the fish intestine contain the most number of bacteria among different species. Some of bacteria that commonly found in aquaculture system were Aeromonas, Escherichia, Enterobacter, Klebsiella, Pseudomonas, Vibrio, Bacillus, Listeria, Staphylococcus, Citrobacter and Edwardsiella. In antibiotic selection had resulted, the differences bacterial genera required the different classes of antibiotic for optimal antibacterial activity.
2.2 Antibiotic and Antibiotic resistant-mechanisms.
Antibiotic or antimicrobial agent can be define as the substances that have capacity to kill or inhibit the growth of microorganisms (Aly & Albutti., 2014). In addition, the antibiotic also used as medical treatment and should be safe to host in body defenses system. Antibiotic reported as used in aquaculture or in livestock production or of interest in human medicine, they were amoxycillin, ampicillin, cephalothin, cephalexin, cefoperazone, ceftiofur, nalidixic acid, oxolinic acid, ciprofloxacin, chloramphenicol, florfenicol, gentamicin, kanamycin, erythromycin, tetracycline, oxytetracycline, sulfamethoxazole, trimethoprim and potentiated sulphonamide (trimethoprimsulfamethoxazole)( Akinbowale et al, 2006). Different antibiotic has its own different chemical structures, and their act on different part of bacterial machinery.
Generally, the antibiotic act by bactericidal effect kills the bacteria by interfering the formation of bacterium cell wall or its cell content. The other is bacteriostatic effect, the antibiotic stops bacteria from multiplying by interfering bacterial protein production (Romero et al., 2012). However, several bacteria may survive under unfavorable
4
condition environm ental changes after selecting mutations that made themselves to improve their fitness for new adaptation. This antibiotic resistant can be categorized into two forms which inherent or intrinsic resistance, the species is not normally susceptible
'
to a particular drugs may be due to the inability of the antibacterial agent to enter the bacteria cell and reach its target site, or a lack of affinity between the antibacterial and its target (site of action), or the absence of the target in the cell. Next, the type acquired resistant represents the major cause for concern because of the transmissible nature of the resistance mechanisms. In this case, the bacterial species is normally susceptible to a particular drug, but some strains express drug resistance. Initially susceptible populations of bacteria become resistant to an antibacterial agent and proliferate and spread under the selective pressure induced by the use of that agent (Romero et al., 2012).
2.3 Disc Diffusion Method
Antimicrobial susceptibility testing is the most often uses the disc diffusion (Kirby- Bauer) due to its simplicity and to the possibility of testing a series of antimicrobial agents in one experiment. According to Tendencia (2004) , the antibiotic- impregnated disk, placed on agar previously inoculated with test bacterium, pick-up moisture and the antibiotic diffuse radially outward through the agar medium which producing an antibiotic concentration gradient. This make concentration of antibiotic at the edge of disk is high gradually will diminishes as the distance from disk increasing to point where it is no longer inhibitory for the organism, which then grow freely. A clear zone or ring is formed around an antibiotic disk after incubation if the agent successful inhibits bacterial growth.
2.4 Enteric Bacteria
Based on Guentzel (1996), enteric bacteria were mostly found or pathogenically at intestinal area of human even animal gut flora. This_bacteria are n;orphologically in rodshaped gram-negative bacteria, in the general clinical the virulence of this bacteria often depends with presence of attachment pili (for hemagglutinating reaction) and sex pili also may be present which it function to transfer genetic information.
Enteric bacteria is under the family of Enterobacteriaceae. It consists of these Genera which they are Escherichia, Salmonella, Shigella, Klebsiella, Serratia, Proteus, Yersinia, Erwinia, Enterobacter, Pasteurellales and others. These bacterial group has their own mechanism in destroying or inhibit their host tract such as triggering inflammatory or disrupt intestinal barrier and absorption occur. Based on studies of Hodges and Gill (2010), the mechanisms for microbial diarrhea by which an organism involve colonize and disrupt intestinal function to cause malabsorption or diarrhea are microbial attachment, localized effacement of the epithelium, production of secretory enterotoxin, production of celldestroying cytotoxin, or direct epithelial cell invasion.
2.5 Shigella spp.
One of Enterobacteriaceae genus known as bacillary dysentery, non-sporulating, facultative anaerobe. Shigella spp. is also a primate-restricted pathogen, which differentiates it from the other members of the Enterobacteriaceae family in which it is classified. Shigella spp. transmission is by the faecal-oral route, and may be via food or water, as well as by person spread such as contaminated hands often contaminate food that is then served to others.Once Shigella bacteria affect their host-microbiota, it known as shigellosis. The Shigella genus is divide into four species Shigella dysenteriae(serogroup A, 15 serotypes),
O
Shigella flexneri ( serogroup B, 19 serotypes), Shigella boydii(serogroup C, 20 serotypes) and Shigella sonnei (serotype D, 1 serotype), these had made divided into multiple serotypes dependent on their O-antigen and biochemical differences (Mattock and Blocker,2017). According to Levine et al. (2013), these four different species linked to disease in varying geographical locations. S, dysenteriae causes severe epidemic in less developed countries, S. flexneri causes disease in developing countries, S, boydii confined in Indian subcontinent and S sonnei occurs in both transitional and developed countries.
Shigella sp. secretes virulence factor that induce inflammation and mediate enterotoxic (ST) effects on the colon, producing the classic watery diarrhea usually seen early in infection. Next, Shigella sp. injects their virulence effectors into epithelial cells via its Type 3 secretion system to subvert the host cell structure and function establishing a replicative niche and causes erratic destruction of colonic epithelium. Then, Shigella sp.
produces effectors to down- regulate inflammation and innate immune response. This eventually promotes infection and limits the adaptive immune response causing the host remain partially susceptible to re-infection.
2.5.1 Shigella sp. in Water
Contamination in water resources is also affected by the water-borne pathogen which included Shigella sp. (Pandey et al., 2014). More than 27% of cases were caused by Shigella sp. In addition, 10.99%, 10.08%, and 6.59% of the cases were caused by Cryptosporidium parvum, Adenovirus 3, and Leptospira, respectively. Nearly 23% and 21 % of the outbreaks were caused by Gastrointestinal Illness (GI) and Shigella spp, respectively. In addition, 16.84%, 12.63%, and 7.37% of the outbreaks were caused by Naegleria fowleri, E.coli0157:H7, and Schistosoma spp., respectively (Craun et al., 2006
and Pandey et al.. 2014).
I
3.0 MATERIALS AND METHODOLOGY 3.1 Sample Collection
The water samples of traditional farm were collected from Kota Samarahan and the other 2 commercial farms from Lundu. The information that can obtained from
management of commercial farm were water quality screening weekly compared to
traditional farm. Commercial farm tend to check water quality as their priorities such as the ammonia contents, dissolved oxygen, pH and water transparency. However, the traditional farmer rarely did the water cleaning and pond treatment. A total of 8 samples within selected four points of ponds as in the Figure 3 .1 were collected from three different aquaculture farm during January 2018. The water samples were collected in 50 ml sterilized falcon tubes from each three different aquaculture farm. After collection, the water samples were kept in thermopol box filled with crushed ice and transported to Microbiology Lab, UNIMAS.
xx
xx
Figure 3.1 The four selected points within I pond
3.2 Enrichment of Water samples
A total of 25 ml of each water samples were inoculated with 225 ml of Buffered Peptone Water (Himedia) and incubated at 37 °C for I 8-24 hours based on research King and Mbewe (2010). After incubation. 1 ml of each sample transferred and supplemented
into 10 ml of selenite cysteine broth (Oxoid) following incubated for 18-24 hours. Ten-fold serial dilution of water samples were performed by aliquots 1 ml of culture dilution into 9 mL of Phosphate Buffered Saline (PBS) with concentration 10, 10°, 19°, 10, 10°. 0.1 mL of each water sample from concentration of 10 °,10, 10° spread onto selective agar Xylose Lactose Deoxycholate (XLD)(Himedia) and the plates were incubated at 37 °C for
18-24 hours.(Saima et al., 2018).
3.3 Isolation of Shigella spp.
A single suspected colony was picked and streaked onto a new XLD agar. Each farm was picked for 20 colonies from previous agar to prepare 20 isolates of the suspected Shigella spp. and incubated for overnight. Each isolate was grow in 5 ml of LB broth and incubated for overnight at 37 °C. After incubation, a total 750 µl of overnight broth mixed with LB broth and glycerol stock and stored in -20 °C for further used.
3.4 Identification of Shigella spp.
3.4.1 Gram Staining
The suspected colony was picked by single loop and transferred into sterilized glass slide. Then, the loop flooded with crystal violet, 1 minute, and washed for 5 seconds, blueviolet appear. The smear was flooded with iodine in 1 minute, rinsed the slide for 5 second and blue-violet still appeared. An addition of decolorized substances which was ethanol. Lastly, the safranin was applied in which the gram negative will tum into pink. All isolates were observed under microscope and gram negative resulted in pink colour.
3.5 Biochemical Characterisation 3.5.1 Oxidase Test
A single loop of isolate was transferred onto an oxidase strip. The result will be represented by the formation of purple color within a few seconds while remain unchanged represented negative oxidase.
3.5.2 Catalase Test
To perform this test, a single loop of isolates was transferred onto a clean glass slide. Then 3 % H202 was added on the slide and mixed together. The presences of bubbles indicated a catalase positive result while absence of bubbles indicated a catalase negative result.
3.5.3 Citrate Test
To run this test, the isolate was inoculated into Simrnon's Citrate Agar (Oxoid), which composed of sodium citrate as carbon source, ammonim dihydrogen phosphate as nitrogen source, and pH indicator, bromothymol blue. The slant agar was incubated at 37 C. The color changed was observed after 24 hours. A color change from green to blue indicated the positive result while a negative result, the color remain unchanged.
3.5.4 Triple Sugar Iron Agar/ KJigler Iron Agar Test
All isolates were characterised using Kligler Iron Agar (Oxoid) to test for glucose, sucrose and lactose fermentation. KIA and TSI tubes are inoculated by stabbing the butt and streaking the surface of the slant. Then, the cap was loosened up to avoid anaerobic environment occurred. After incubation for 18-24 hours at 35° C-37C, the slants are observed for reactions typical of Shigella characteristic. (Ajello et al., 2003).
I 0
3.6 Molecular Characterization 3.6.1 Genomic DNA Extraction
\
DNA extraction was prepared by boiling cell method based on Macias et al (2007).1 ml of culture from overnight LB broth were centrifuged (Hettich Mikro 22 R) for
.
3 minutes at 12,000 rpm. Then, the supernatant was discarded and the left pellet was suspended with 400 l of sterile dH,O. The suspended pellet was put in water bath for 10 minutes at 100 °C and cooled on ice for 10 minutes. After that, it was centrifuged (Hettich Mikro 22 R) for 5 minutes at l 0,000 rpm and the supernatant was transferred into new microcentrifuge tube. The samples were kept in freezer for further use.
3.6.2 Polymerase Chain Reaction
The PCR reagent in a final volume of 25 l based on Amani et al., (2015). The PCR mixture as mentioned as Table 3.6.2.2 and thermal cycling of amplification mixture was performed in 30 cycles (Eppendorf).The PCR programme as listed in Table 3.6.2.3 for stx gene. The PCR products were electrophosed in 1 % of agarose, 500 mA, 90 V for 1 hour 30 minutes followed by staining with ethidium bromide (Amresco X328-10ML) then visualized under ultraviolet light.
Table 3.6.2.1 The stx forward and reverse primers sequences
Primer Sequences
Stx- Forward '5-TGG AAA AAC TCA GTG CCT CT-3'
Stx- Reverse '5-CCA GTC CGT AAA TTC ATT CT-3'
II
Table 3.6.2.2 The master mixtures for stx amplification
Reagent lX of Reaction, pl
IO X PCR Buffer 2.5 ,
-
dNTP mixtures 1.0
stx(forward) 1.0
stx(reverse) 1.0
50mMMgC12 1.5
Tag Polymerase 0.5
DNA template 1.0
Distilled water 16.5
Total volume 25.0
Table 3.6.2.3 The PCR programme for stx gene
PCR Programme Temperature, 'C Time
Pre-denature 94 3 minutes
Denaturing 94 45 seconds
Annealing 59 45 seconds
Extension 72 1 minute
Final Extension 72 5 minutes
12
3.7 Antibiotic Susceptibility Tests
Three bacterial isolates which represented each three aquaculture were analysed for their susceptibility towards different antibiotics by disk diffusion method. Based on the previous studies by Kathleen et al. (2016), the bacterial isolates were adjusted to 0.5 McFarland's turbidity standard. After reached the right turbidity, a sterilized swab was dipped into culture broth and swabbed onto the surface of Mueller-Hinton Agar (MHA).The culture was assessed with tetracycline, sulphonamides, ampicillin, erythromycin and trimethoprim/ sulfamethoxazole. The antibiotics disc were embedded on MH agar. After 16-18 hours of incubation at 3 5°C, results will be interpreted as either sensitive, intermediate, or resistant according to the inhibitory zone diameters around the disks using CLSI breakpoints.
I5
