Embed Size (px)
Citation preview
The Incidence of Vibrio parahaemolyticus in Raw Vegetables at Local Wet Markets and Supermarkets in Kuching Sarawak
Sabatini Jihob
Bachelor of Science with Honours QR 82 (Resource Biotechnology) S6 2011 SIl3 2011
nnlt Khhlmlt MIJdmiddotr middot t ltkadlmiJ UNMISm MALUmiddots MRAWAK
The Incidence of Vibrio parailaellloiy tictls in Ra Vegetables at Local Wet Markets and Supermarkets in Kuching Sarawak
Sabatini l ihob (22218)
This project is submitted in pal1 ial fulfilment 0 the requirements for the degree of Bachelor
and Science with Honours (Reso urce Biotechnology)
Faculty of Resource Science and Techno logy
Un iversit i Malaysia Sarawak
2011
ACKNOWLpoundDCE~1ENT
Thanks be to God His grace and blessings had guided me to the completi on of thi s fin al year
project and a ll along my 3 years of studying in UNIMAS He is whom ltumed to in times of
weary and confusion By His will this project was finall y completed
First and forem ost I would li ke to express my deepest gra titud e and appreciations to
my respec ted supervisor Dr Lesley Maurice Bi lung for her endless eff0l1 advice suggesti on
and supervision I am also very grateful to have Dr Micky Vincent as my co -supervisor who
has been so helpfu l Not forge tting thanks a lott o Dr Samu el Lihan who assis ted in the fin al
editing of this writing
My gratefulness is a lso extended to a ll the postgraduate students in Microb iology Jab
especially Vel nett i and Ka thl een for their countless help They have been so generous in
sharing their know ledge and never fa iled to lend a helping hand in my times of d ifficulties
This project wouldn t have been a success wi tho ut the both of them tha nks and all the best
for the future l My thankfulness also reached ou t to all m y lab mates Mim Ard i Lazarus
Sheila Tata ng Matthew and the others for being such wonderful considerate and help[d
fr iends My hea11felt apprec ia tion also goes to my lovely parents sib lings fri ends and
beloved ones fo r the ir unfai ling love cons istent prayers and constant suppol1 Last but not
least thank you to a ll my lecturers course mates nnd everyone that has con tributed eit her
direc tly or ind irectl y tow ards the completion of this project
)
-~-- l
DECLARATION
I hereby declare thnt this Fina l Year Project report is based on m y origina l work except for
quotations and citations which have been properly acknowledged [ also declare that it has not
been previously or concurrentl y subm illed fo r any other degree in UNIMAS or other in stitut ions
SABATINI JU- OB (222 18)
Date 305 gt01
11
~
bullbull
TABLE OF CONTENTS
ACKN OWLEI)GEMENT
DECLARATJO II
TABLE OF CONTENTS I II
LIST OF ABBREVIATIONS v
LIST OF TABLES D FIGURES V I
ABSTRACT I ABSTRAK
CHAPTER 1
CHAPTER 2
CH PTER3
INTRODUCTION 2
Lrt ERATlJRE REVIEWS 5
21 The genus Vibrio 5
22 Vibrio parahaemolylicus 5
23 Fresh vegetables 7
24 Clinica l cha ract eristics of V parahaemolyticus 8
infections
25 Iso lation of V parahaemolyticus 9
26 Id entilication through Gra m-Stain and biochemical 10
tes ts
JllATERlAL AND METHODS 13
31 Samples co ll ection 13
32 Samples preparat ion and enrichment 14
III
__a ------ _bull 1 shy
33 Isolation o f V parahaemolylicus 14
34 Preparation for Gram-stain and biochemical tests 15
35 G ram slain prolocol 16
36 Biochemicallesls 16
36 1 Voges-Proskauer test 18
362 Methyl-Red test 18
363 Sa lt tolerance test IS
36 4 Triple sugar iron test (TSI) 19
365 Hyd rogen sulfid e and gas production 19
366 Ci trate test 19
RESULTS 20
41 Iso lation of V parahaemolyticlIs 20
43 Gram-stai n and biochemical tests 23
CHAPTER 5 DI CUSSION 28
51 Vegetab les types 28
52 Co loni es appearance on selective aga r 28
53 Isolation rate of V paraiaemolylicus on TCa and 30
C HROMagar Vibrio
54 In terpre ta tions of biochemical tests 34
55 Preva lence of V parahaemolylicus 35
CIIAPTER 6 CONCLUS ION 37
CHAPTER 7 REFERENCES 39
IV
-shy
--
C
APW
g
KJA
NK
1111
mm
MR
NaCI
PCR
Spp
TeBS
TSI
VP
V parahaemoLytiCLIs Vp
WHO
LIST OF ABBREVIATIONS
percentage
degree celcius
Alkaline Peptone Water
gram
hyd rogen sulfite
alkaline slant acidic bUlt
acidic slant alkaline butt
microliter
milli liter
milimeter
Methyl-red
Sodium chloride
Polymerase Chain Reaction
species
Tiosulphate Citrate Bile Salt Sucrose
Tliple Sugar Iron
V oges-Proskaeur
Vibrio parallOemolyticlIs
World Heal th Organizati ons
v
~-
LIST OF TABLES
Tables Page
Table 21 Standard biochemical test reactio ns of V parahaemolyliclIs 12
Table 31 Types of raw vegetable samples collected from wet markets and 13
supermarkets
Table 41 Results for the presence of V parahaemolyticus on TCBS and 2 1
CHROMa gar Vibrio from the vegetable sa mples
Table 42 Result s of microscopic observations of the suspected V 27
parahaemolylicus iso lates
Table 43 Overall resuit of biochemical tests 24
vi
--bull i~
LIST OF FIG URES
Figures Page
Figurc 2t SEM observation o f Ii parahaemolyticus Image source Belas amp 6
Colwell (1 982)
and C I-IR OM agar Vib rio trade based on different sam pling locr io ns
Figure 31 Biochemical keys fo r the identification o f Vibrio species 17
Figure 41 The isolation o f Ii parQhaemolyticus on TCBS 22
Figure 42 Appearance of mixed colonies on CHROM agar Vib riotrade 22
Figure 43 Pure mauve co lonies obtained a fter sub -culturing 23
figurc 44 Reac tio ns of (a) B7KBS and (b) B8CW towards Voges -Proskauer test 24
Figure 45 Reactio ns of (a) B7KBS and (b) BI CW towards M dhly-red test 25
Figure 46 The reaction of a ll isolates towards citrate test 25
Figure 47 The reaction o f (a) B7KBS = KJA and (b) B3KW = AK fo r TSltes t 26
Figure 48 The reactio n o fB7KBS towards (a) 0 NaC I and (b) 8 Nae 27
Figu re 5 1 Bar graph o f the di stribution of presumptive pas iti ve sampl es on TCBS 30
Figure 52 Pie chart o f the distribu tion of presumptive Ii paraliaemolyliclls 31
between different vegetable types on CHROM aga r Vibrio
Figure 53 Pi e chart of the distribution of presumptive Ii parahaemolylicus 33
between di fferent vegetab le types on CHROMagar Vibriotrade
VII
--~bull i lj
Tbe Incidence of Vibrio p arahaellloly ticlis in Raw Vegetables at Local Wet Markets and Supermarkets in Kuching Sarawak
Sabatini lihob
Resource BIOIClhnoltlg) Progrtlm me Facu Ity or Resource Science Bud T~lln(llogy
Uni versity Malaysb Sa rawa k
ABSTRACT
Vibrio parawemoyliCLls is a foodbome pa thogen that is regular ly assoc iated with the consu mption o f
raw or undercooked seafood The mos t frequent sympto m of V parahaemoylicus infecr ion is
gastroenteritis Recen tly this pa thogen was also disco vered in raw vegetable samples in Peninsular
Malaysia Thus the aim of this study is to isolate and de tect V paahaemoylicu~ in raw vegdables T he samples which consisted of carrot fourmiddot winged bean cabbage and bea n sp ro ul were purc hased
tiom two wet markets and two supenn arkets in Kucll ing Sarawak In th is stud y alkaline peplone
water (A PW) wa s used as the enridunent medium The combinati o n o f two types o f selective agar
TCBS and CHROMa gar Vibrio nbull1 was successfully app lied to isolate the bacteria A fte r a series of
biochemical tests only 2 out of the 80 samples were pos itive for the p resence of V parahaemoylicLls
Although the peroentage of V pOrehaemolylics prevalence was low (2 5) the resu lts h ighli ghted
the fac t that raw vege tables could also be contaminated w It h V parahaenro~Vlicus
Key words Vib rio parahemotyfic~ raw vegetables TC BS and CHROMagar Vibrio rM
ABSTRAK
Vibrio parawcmoylicus merupakan sejenis patogen bmvaan makanafl )long sering dikairkan dengan
pengambiall makonon laul seewa men fa h Tonda paling kemp bagi j angkitall k parahwnJ r lvticlfs
ialah gasroenlritis TerbolU pafogen ini Ina dijumpa i di dalam sUlllp~1 scJyuran melJfah di
Semenanjung A1nlaYiia Kajial1 ini berlujuan un uk mengasmg dan mengesan V nanlhaemolliqi di
daall1 sampel $oyuran menfah Sampel yang lerdiri doripada obok merah kac(mg botol kobis dan
(l uge teah dlbeli dari dU(J pasai basah Clnd clua pasar raya di Kuchi lg Sorm lak Il l pepton beralktrli
(A PW) fel(lh digul1akan Ill1tuk memperbonyakko1 jwn Jah bakfcna Kombilw si pelggunaon agar
terpilih Tess dan CHROkiagar Vibrio T1 tdoh bejaya mel11ellcilkon ba( ur ia yallg dikehendoki
Selelah beberapa siri lljion biokimia didapafi 2 daripada 80 sampel relah dlcemQj oleh 1 rar(fhoenlJliJji(u~ Mcsklpun perafus kehadiran V norahaemolvticus adalah remJah (25~ o)
kepuflfsan kajian ini menunjukkan bahawo saYllran mental boleh dicemari ole Y paraha~moh IIcm
Kata klllt ci Vibrio oorqhaemol liclfs sayuran men fah TCBS dOll CHROtviagar Vihriotrade
-
CHAPTER 1
INTRODUCTION
11 Introduction
Foodbome illness assoc iated with consumption of contam inated food is a corrunon hea lth
problem that occurs worldwide The global inc idence of food bo rne ill ness is d ifficu lt to
estimate but it has been reported that in 2005 alone around 76 million cases of food bo rne
diseases resu lting in 325000 hospita liza tions and 5000 dea ths are es timated to occur in the
United State (WHO 20 11 ) In addition food contaminat ion also causes enormous socia l and
economical burden on the community A consumer research repon released on 2010 revea led
that the healt h-related costs of food-borne illnesses had cost up to 152 billion do llar a year
includ ing the costs of med ica l bills lost wages and lost produc tivi ty (Smii middot 2010) Thus
food safety is cun-entl y an increasing important pub lic health issue laquougovenunents all over
the world are intensify ing the ir efforts to improve food safety
Among the majo r foodbome illness caused by mi croorgan ism are Salmonellosis
Campylobacteriosis Listeriosis and infectio ns due to entero haemonha gic (caJing intes tinal
bleeding) E coli (WHO 2007) III recent years Vibriosis had emerged to be an impOItant
bac te rial disease that canses human intestinal and ext ra-intestinal infections a ll over the world
(Chan elol 2002 Garc ia el al 2009 and Harth et al 2009) The three Vibrio species o f
primary significance in foodbo rne illnesses are V cholerae (serogroups 01 non-Ol and
2
- bull i
0 139) V vulnijiclIs and V paralwemolyricus Presently gast roenteitis caused by V
parahaemolyticus is extensively documented due to its increasing outbreaks (Alam el 01
2003 Gwendelynne el 01 2005 Gorny 2006 Harth et 01 2009 Roque et 01 2009)
V parahaemolyriclls is widely present in brackish and marine waters and infection to
human is regula rl y associated with the consumption of contaminated raw or undercooked
scafood (Guoxia ng el 01 2009) Ea rli er studies also demonstrated that V parahoemolylicus
can be easily iso lated from costal water sediments suspended particles and plankton due to
its halophi lic characteristic (Marlina el 01 2007 Zinuneman el 01 2007 Zu lkifli el al
2009 Vimala et 01 2010) However recent foodbome outbreaks throughout the world have
been intensive ly linked to consu mption of fresh fruits vegetables and unpasteurized juices
(Gorny 2006) Most recently Tunu ng et 01 (20 10) had rep0l1ed th at the vegetable samples
from supermarkets and wet markets in Selangor Malaysia were posit ive for pathogeni c
species of V parahaemolylicls Conversely there is no ot her known documented research on
the prevalence of this species in vegetables
In this st udy vegetables from two wet markets and two supem1arkets in Kuching
Sa rawak were sampled for the presence of V parallOemolyliclis The sa moles underwent
selective enrichment with alkaline peptone water (APW) supp lemented with 3 NaCI Then
presumptive V parahaemolYlicus were iso lated using TeES agar and CH ROMagar VibrioT
Following isolat ion gram stai ning together with a series of biochemica l tests were perfonned
to assist in the identification and confirmation of the iso lates
3
- bull i ~
IZ Objectives
This study was underta ken with the fo llowing objecti ves
I To isolate presumptive V parahaemolYlicus from marketed vegetabl es samples
in Kuching Sarawak by using Te BS and CHROMagar VibrioTM
II To confirm th e suspected V parahaemolyficus iso lates from marketed
vegetables samples in Kuching Sarawak by usi ng Gram stain and biochemical
tests
4
- bull i ~
USI KhJtlmal Makumat U umk VMVl~m ItLAYSlA AAAWAK
CHAPTER 2
LITERATURE REVIEW
21 The genus Vibrio
The genus Vibrio belongs to the fami ly Vibrionaceae and it consists of at least 34 recogn ized
species (HPA 2007a) In general Vibrio spp are Gram-negat ive fac ulta tively anaerobic
mOlile curved rods bacteria with a single polar flagellum (FEHD 2005) In surface waters of
the world Vibrios are among the mos t common surface organisms (Adeleye et aI 20 J 0)
They are present in both marine and fresh wa ter habiwts and commonly associa ted with
aquatic animals Vibrio pp such as V choerae V parahaemoylicus V vu nificus V
aginoylieus V mimicus V luvia lis V filmissii V melselmikovii V tollisae and V
dalltsea are common human pathogens (Tison 1999 Fi nkelstein 2000) T hey account for a
significant proportion of human infections as most of these vibrios secrete enterotoxins to
foods water or in the gastrointestinal tract (Nishibuchi and DePao la 2005)
22 Vibrio paralJlell1oytictls
Vibrio parahaemoy licus which is formerly known as Pasteurella parahaeliloytlca is a
non-sporing Gram-negat ive facu ltatively-anaerobic bacterium (Curti s el 01 2003) This
5
- ~
halophilic bacterium inhabits warm estuarine waters world wide (Bilung el al 2005) Thus il
reqlllres salt to survive and appears in high er concentrations during the warmer summer
months
The optimum temperature fo r the grow th of V parahaemolylicus is 37 degC and at Ihis
optimum temperature the bac terium has generation times of8 to 9 minutes in water and 12 [ 0
18 minutes in seafood (Montville and Matthews 2008) Thi s bacterium is the most IKeiy
Vibrio species to be imp licated in food borne diseases although both V vulnificus and V
cholera may also causes food bom e infections (Lawley e l al 2008) Clinical isolates of V
parrhaemolyticus produce beta type hemolysis on blood aga r (Wagatsuma agar) The
hemolysis is ca ll ed the Kanagawa-phenomenon (KP) which is associated with the production
ofa thermo stabl e d irect hemolys inTOH (Boyd 2008) TOH caused damage to the euka ryotic
cells by acting as a pore fonning toxin that alters the ion balance of cells (Boyd 2008)
Figure 21 SEM observa tlOos of V plrh cm)JJ(cu~
Image source Belas amp Colwe ll ( 1982)
6
-- UI
23 Fresh vegetables
Consumpti on of raw vegetables is common throughout the world not onl y owing to Ihe health
benefit but they are thought to be free from disease causing pathogens However recent food
borne outbreaks worldwi de is associ ated to consumption of fresh frui ts uncooked vegetables
and unpasteurized juices (Gorny 2006) Fruits and vegetable especially those that are eaten
raw without peeling can be the vehicles for transmission of a wide range of mi croorganis m
(Erdogru l and Sener 2005)
Based on Haeton and Jones (2008) E coli Salmonella spp and Listeria spp are
commonly present in vegetable samples Meldrum el 01 (2009) report ed that the two recent
large ou tbreaks in the United Kingdom showed signi fi cant health problems that cou ld ari se
from consumption of contaminated salad Another study of the preva lence of pathogens in
vegetables was reported by Little el 01 (2007) in which they found Listeria monocylOgenes in
ready-to-eat mixed salads in the United Kingdom Several other studi es on d ifferent
microorga nism isolated from vegetabl es were also report ed recent ly by local researchers
(Chai el 01 2008 Han el 01 2009 Ponni ah el 0 2009)
In Mala ys ia the presence of V parahoemo yticus has also been repOIt ed in raw
vegetables samples from wet markets and supeLl11arkets in Selangor (Tunung et 0 20 I 0)
This same study discovered that raw vegetabl es such as OenGnthe st l7i(era (Japanese
parsley) and C[cumis sali vus (Cucumber) were contaminated with virulent V
7
parahaemoiYlicus Consequen tly these vegetables could act as a transmission route and
poses risk to consumers after consumption Tunung el 01 (2011) had also demonstrated that
the vegetable samples fro m wet market contained higher levels of V paraiJaemolylicls as
compared w ith supennarkets The research suggested that there are many factors that can
contri bu te to the distribution of V parahaemoiylicus such as temperature holding ti me as
well as hygiene and sanitation practice (Tunung el aI 2011) However there is no known
documented research on V parahaemolYlicus in raw vegetables in Sarawak
24 Clinical characteristic of V parahaelllolyliclis infections
II parahaemoiyticus causes three distinct illness syndromes which are gastroenteritis wound
or soft tissues infections and septicemia (Sillljee 2007) The most freq uen t syndrome is
gastroeuteritis The symptoms include dianhea with abdomina l cramps nausea vo miting
headache and low-grade fever (S tarlin 2005 WilU1 and Konema n 2006 Montville and
Matthew 2008) Sometimes the diarrhea is bloody w ith stools described as meat washed
since tl1e stool is reddish and watery (Qadri el aI 2009)
As for wound or tis sue in fections this coudition occurs by the exposure of broken skin
or wound to warm seawater and seafood dripp ings (Ralph and Currie 2007 Simjee 2007)
In severe cases serious wound infection will resu lt in necrotizing fasc iiti s (Ralph and Curri e
2007) Although less common in V paraiwemolyliclIs infection septicemia is characteri7cd
by fever or hypotension with the isolation of V parahaemolyticus in the blood Septicemia
typicall y occurs in person with underlying health problems such as liver disease diabetes
cancer or other immune disorder (Simjee 2007) V parahaemoyticlis can even ca use fala l
septicemia in an immune-compromised hosts
The symptoms usuall y occur on the average of 15 hours but can began as early as 4
hours and as late as 36 hours after exposure and this will continue up to 3 days (Barceloux
200S) However these illnesses are usua lly categorized as a short illnesses and no prolonged
enect will take place after recovery (Wright et al 2009)
25 Isolation of V parahael1lolyliclIs
For the isolation of V parahaemoyticus an initial emichment using alka line peptone wat er
(APW) with pH S5 would be useful as this medium supports the growth uf viable ce lls
(Simjee 2007) Plati ng of emichment broth onto TCBS agar has been widely used for
selective isolation of V parahaemolytics from foods (Thompson el ai 2006 Oi Pinto et al
201 0 Canizalez -Roman et ai 2011 ) An average of 24 hours incubation at 37 degC is requ ired
fo r typica l V parahaemolylicus to develop colonies that are smooth gree n (s u~rose nega tive)
and 2 to 3 nun in di ameter on TCBS (ISO 2007) However V parahaenlOlytlcus co lonies on
TCBS agar are difficult to distinguish vi suall y from other bacterial colonies since they can be
covered by yellow color produced by sucrose-fennenting bacteria (Thompson el 01 2006
Rambach 2007)
9
_ ------~-- i ~~
Another widely used selective medium is CHROMagar VibrioT On this medium V
pllrahaemoiy liclis will developed mau ve color colonies (Kudo et aI 2001 Oi Pi nto 2010
Canizalez-Roman 2011) This agar medium contains substrates for beta-galactosidase which
was developed specifically to differentiate V parahaemolytics from ot her bacteria by us ing a
chromogenic su bstrate instead of sugar fermentation in traditional growth med ia such as
TCBS (Hara-Kudo et 01 2001) Therefore it is more sensitiv e and accurate for identi fying V
porahllemolyticlIs in food samples rather than the use of Te ES (Oi Pinto 2010 Canizalez-
Roman 20 II)
26 Identification through Gram-stain and biochemical tests
V parahaemoiyticus is a Gram-negative bacterium th us this organi sm stained pink or red
after Gram staining (HPA 20073) Under microscopic observation a typical V
pllrahaemolyticus appeared as rod characterist ica lly curved or comma-shoped The bacterium
is also positi ve for motility under microscopic observations (ISO 2007 BPA 2007a)
V parahaemolylieus ca n be identified through a seri es of biochemical tests According
to Khan et 01 (2007) V para haemolyticus can be distinct ively differentia ted fro m the other
Vibrio spp by performing sa lt tolerance and acid fro m femlen tation tests A typical V
parahaemoiYlicus co lonies would not grow in AFW without th e presence of at least 3 of sali
10
~-------
but it can only endure the presence of salt up to 8 (ISO 2007 Khan el aI 2007) In the
other hand the fermentation test for acid production is the most important test to differentiate
between V parahaemolyliclIs and V vulnificus species (Khan el aI 2007) Jayasinge et ai
(2010) even suggested that the combination of salt toleranc e test Voges-Proskauer test and
ONPG test can provide a strai ghtforward differentiation of V parahaemoly liclis from the
other Vibrionaceae species (Figure 31)
Citrate test screens the ab ilit y of bacterial isolates to utilize ci trate as its carbon and
energy source (MacFaddin 2000) This test is a very important test to identify gram-negative
pathogens (Tang el aI 1998) The MR-VP medium which was used for Met hyl red and
Voges Proskauer test contains glucose and peptone Methyl red test screens for the bacterial
metaboli sm pathways for glucose fermentation while the Voges-Proskauer tes t is an assay for
organisms which ferment glucose with only one fermentation product usuall y acetic acid
The TS[ test is another useful test tha t can distinguish V parahaemolyticus from other
bacteria (ISO 2007) The TSJ agar med ium contains peptone fenous sulfate lactose sucrose
and a small amount of glucose (dex trose) The pH indi cator is phenol red This medium
differentiates bacteria based on their ability to fennent glucose lactose ando r sucrose and on
their abi lity to reduce sulfur to hydrogen sulfide (H S) The T SJ test also haj the abi lity to
determine the production of H]S (Kim et aI 1999)
In additi on other bi ochemica l tests such as oxidase lysine decarboxylase om ithi ne
decarboxylase ca talase and indole can also be used for the purpose of identifying V
II
-- ---- - ----------- - ~i
parahaemoyticus iso lates (ISO 2007 Jayasinghe et ai 2007 Khan et aI 2007) The
biochemical reactions o f V parahaemoiyticus are summarized in Table 21
Table 21 Siandard biochemical tesl reactions of V porahaemoyticus
TeSi S Reaction of V parahaem olyaclIs Refe rences Salt tol ance
+
Sali ne TSI agar KIA
No HS and gas production
Simmons Citrnh
Voges-Proskauer
Methyl-Red +
[SO (2007)
Jayasinge et al (2007)
Khan et of (2007)
ISO (2007)
Kim et I (1999)
Abide et til (1999)
Kim 11111 (1999)
Khan et til (2007)
Jayasinge el III (20 (0)
Kim et 0 (2 007)
Note (-) ~ nega live (+) ~ positi ve K ~ alka line ~ aC idic
12
CHAPT ER 3
MATERIALS AND METI IODS
31 Samples collection
Eighty raw vegetable samples were collected week ly from two wet markets (WI amp W2) and
two supennarkets (S 1 amp S2) in Kuching Sarawa k The collected samples consisted o f four
di fferent groups of vegetables which are regularly consumed ra w by the locals These samples
wcr~ rand omly selected within ten weeks -period which is from October 20 10 to January
20 11 Duri ng collection each sample was transferred to clean plastic bags labeled acco rding
to the place they were collected and were analyzed immed iately on arriva l to the labo rat ory
The types of raw vegetable sa mples obtained were as shown in Table 31
Tab le 31 Types o f raw vegetable samples co llected from wet markes and supcmlarkels
Group Loca l nam e English nam e Scienti fi c Home l mple coding No or samples
Soil Kubis Cabbage Brassica v eracea K 20 Roots Lobak Canmiddotot Douclfs carola C ~O
mera h Beans KaCtlO g Four-winged PsophOCaJjJ lls KB cO
bow l bean JeIlagulIolobus Shoot Tauge Bean-I_prout Vigna rad mc T 10 Total 80
13
---------~ I I
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
nnlt Khhlmlt MIJdmiddotr middot t ltkadlmiJ UNMISm MALUmiddots MRAWAK
The Incidence of Vibrio parailaellloiy tictls in Ra Vegetables at Local Wet Markets and Supermarkets in Kuching Sarawak
Sabatini l ihob (22218)
This project is submitted in pal1 ial fulfilment 0 the requirements for the degree of Bachelor
and Science with Honours (Reso urce Biotechnology)
Faculty of Resource Science and Techno logy
Un iversit i Malaysia Sarawak
2011
ACKNOWLpoundDCE~1ENT
Thanks be to God His grace and blessings had guided me to the completi on of thi s fin al year
project and a ll along my 3 years of studying in UNIMAS He is whom ltumed to in times of
weary and confusion By His will this project was finall y completed
First and forem ost I would li ke to express my deepest gra titud e and appreciations to
my respec ted supervisor Dr Lesley Maurice Bi lung for her endless eff0l1 advice suggesti on
and supervision I am also very grateful to have Dr Micky Vincent as my co -supervisor who
has been so helpfu l Not forge tting thanks a lott o Dr Samu el Lihan who assis ted in the fin al
editing of this writing
My gratefulness is a lso extended to a ll the postgraduate students in Microb iology Jab
especially Vel nett i and Ka thl een for their countless help They have been so generous in
sharing their know ledge and never fa iled to lend a helping hand in my times of d ifficulties
This project wouldn t have been a success wi tho ut the both of them tha nks and all the best
for the future l My thankfulness also reached ou t to all m y lab mates Mim Ard i Lazarus
Sheila Tata ng Matthew and the others for being such wonderful considerate and help[d
fr iends My hea11felt apprec ia tion also goes to my lovely parents sib lings fri ends and
beloved ones fo r the ir unfai ling love cons istent prayers and constant suppol1 Last but not
least thank you to a ll my lecturers course mates nnd everyone that has con tributed eit her
direc tly or ind irectl y tow ards the completion of this project
)
-~-- l
DECLARATION
I hereby declare thnt this Fina l Year Project report is based on m y origina l work except for
quotations and citations which have been properly acknowledged [ also declare that it has not
been previously or concurrentl y subm illed fo r any other degree in UNIMAS or other in stitut ions
SABATINI JU- OB (222 18)
Date 305 gt01
11
~
bullbull
TABLE OF CONTENTS
ACKN OWLEI)GEMENT
DECLARATJO II
TABLE OF CONTENTS I II
LIST OF ABBREVIATIONS v
LIST OF TABLES D FIGURES V I
ABSTRACT I ABSTRAK
CHAPTER 1
CHAPTER 2
CH PTER3
INTRODUCTION 2
Lrt ERATlJRE REVIEWS 5
21 The genus Vibrio 5
22 Vibrio parahaemolylicus 5
23 Fresh vegetables 7
24 Clinica l cha ract eristics of V parahaemolyticus 8
infections
25 Iso lation of V parahaemolyticus 9
26 Id entilication through Gra m-Stain and biochemical 10
tes ts
JllATERlAL AND METHODS 13
31 Samples co ll ection 13
32 Samples preparat ion and enrichment 14
III
__a ------ _bull 1 shy
33 Isolation o f V parahaemolylicus 14
34 Preparation for Gram-stain and biochemical tests 15
35 G ram slain prolocol 16
36 Biochemicallesls 16
36 1 Voges-Proskauer test 18
362 Methyl-Red test 18
363 Sa lt tolerance test IS
36 4 Triple sugar iron test (TSI) 19
365 Hyd rogen sulfid e and gas production 19
366 Ci trate test 19
RESULTS 20
41 Iso lation of V parahaemolyticlIs 20
43 Gram-stai n and biochemical tests 23
CHAPTER 5 DI CUSSION 28
51 Vegetab les types 28
52 Co loni es appearance on selective aga r 28
53 Isolation rate of V paraiaemolylicus on TCa and 30
C HROMagar Vibrio
54 In terpre ta tions of biochemical tests 34
55 Preva lence of V parahaemolylicus 35
CIIAPTER 6 CONCLUS ION 37
CHAPTER 7 REFERENCES 39
IV
-shy
--
C
APW
g
KJA
NK
1111
mm
MR
NaCI
PCR
Spp
TeBS
TSI
VP
V parahaemoLytiCLIs Vp
WHO
LIST OF ABBREVIATIONS
percentage
degree celcius
Alkaline Peptone Water
gram
hyd rogen sulfite
alkaline slant acidic bUlt
acidic slant alkaline butt
microliter
milli liter
milimeter
Methyl-red
Sodium chloride
Polymerase Chain Reaction
species
Tiosulphate Citrate Bile Salt Sucrose
Tliple Sugar Iron
V oges-Proskaeur
Vibrio parallOemolyticlIs
World Heal th Organizati ons
v
~-
LIST OF TABLES
Tables Page
Table 21 Standard biochemical test reactio ns of V parahaemolyliclIs 12
Table 31 Types of raw vegetable samples collected from wet markets and 13
supermarkets
Table 41 Results for the presence of V parahaemolyticus on TCBS and 2 1
CHROMa gar Vibrio from the vegetable sa mples
Table 42 Result s of microscopic observations of the suspected V 27
parahaemolylicus iso lates
Table 43 Overall resuit of biochemical tests 24
vi
--bull i~
LIST OF FIG URES
Figures Page
Figurc 2t SEM observation o f Ii parahaemolyticus Image source Belas amp 6
Colwell (1 982)
and C I-IR OM agar Vib rio trade based on different sam pling locr io ns
Figure 31 Biochemical keys fo r the identification o f Vibrio species 17
Figure 41 The isolation o f Ii parQhaemolyticus on TCBS 22
Figure 42 Appearance of mixed colonies on CHROM agar Vib riotrade 22
Figure 43 Pure mauve co lonies obtained a fter sub -culturing 23
figurc 44 Reac tio ns of (a) B7KBS and (b) B8CW towards Voges -Proskauer test 24
Figure 45 Reactio ns of (a) B7KBS and (b) BI CW towards M dhly-red test 25
Figure 46 The reaction of a ll isolates towards citrate test 25
Figure 47 The reaction o f (a) B7KBS = KJA and (b) B3KW = AK fo r TSltes t 26
Figure 48 The reactio n o fB7KBS towards (a) 0 NaC I and (b) 8 Nae 27
Figu re 5 1 Bar graph o f the di stribution of presumptive pas iti ve sampl es on TCBS 30
Figure 52 Pie chart o f the distribu tion of presumptive Ii paraliaemolyliclls 31
between different vegetable types on CHROM aga r Vibrio
Figure 53 Pi e chart of the distribution of presumptive Ii parahaemolylicus 33
between di fferent vegetab le types on CHROMagar Vibriotrade
VII
--~bull i lj
Tbe Incidence of Vibrio p arahaellloly ticlis in Raw Vegetables at Local Wet Markets and Supermarkets in Kuching Sarawak
Sabatini lihob
Resource BIOIClhnoltlg) Progrtlm me Facu Ity or Resource Science Bud T~lln(llogy
Uni versity Malaysb Sa rawa k
ABSTRACT
Vibrio parawemoyliCLls is a foodbome pa thogen that is regular ly assoc iated with the consu mption o f
raw or undercooked seafood The mos t frequent sympto m of V parahaemoylicus infecr ion is
gastroenteritis Recen tly this pa thogen was also disco vered in raw vegetable samples in Peninsular
Malaysia Thus the aim of this study is to isolate and de tect V paahaemoylicu~ in raw vegdables T he samples which consisted of carrot fourmiddot winged bean cabbage and bea n sp ro ul were purc hased
tiom two wet markets and two supenn arkets in Kucll ing Sarawak In th is stud y alkaline peplone
water (A PW) wa s used as the enridunent medium The combinati o n o f two types o f selective agar
TCBS and CHROMa gar Vibrio nbull1 was successfully app lied to isolate the bacteria A fte r a series of
biochemical tests only 2 out of the 80 samples were pos itive for the p resence of V parahaemoylicLls
Although the peroentage of V pOrehaemolylics prevalence was low (2 5) the resu lts h ighli ghted
the fac t that raw vege tables could also be contaminated w It h V parahaenro~Vlicus
Key words Vib rio parahemotyfic~ raw vegetables TC BS and CHROMagar Vibrio rM
ABSTRAK
Vibrio parawcmoylicus merupakan sejenis patogen bmvaan makanafl )long sering dikairkan dengan
pengambiall makonon laul seewa men fa h Tonda paling kemp bagi j angkitall k parahwnJ r lvticlfs
ialah gasroenlritis TerbolU pafogen ini Ina dijumpa i di dalam sUlllp~1 scJyuran melJfah di
Semenanjung A1nlaYiia Kajial1 ini berlujuan un uk mengasmg dan mengesan V nanlhaemolliqi di
daall1 sampel $oyuran menfah Sampel yang lerdiri doripada obok merah kac(mg botol kobis dan
(l uge teah dlbeli dari dU(J pasai basah Clnd clua pasar raya di Kuchi lg Sorm lak Il l pepton beralktrli
(A PW) fel(lh digul1akan Ill1tuk memperbonyakko1 jwn Jah bakfcna Kombilw si pelggunaon agar
terpilih Tess dan CHROkiagar Vibrio T1 tdoh bejaya mel11ellcilkon ba( ur ia yallg dikehendoki
Selelah beberapa siri lljion biokimia didapafi 2 daripada 80 sampel relah dlcemQj oleh 1 rar(fhoenlJliJji(u~ Mcsklpun perafus kehadiran V norahaemolvticus adalah remJah (25~ o)
kepuflfsan kajian ini menunjukkan bahawo saYllran mental boleh dicemari ole Y paraha~moh IIcm
Kata klllt ci Vibrio oorqhaemol liclfs sayuran men fah TCBS dOll CHROtviagar Vihriotrade
-
CHAPTER 1
INTRODUCTION
11 Introduction
Foodbome illness assoc iated with consumption of contam inated food is a corrunon hea lth
problem that occurs worldwide The global inc idence of food bo rne ill ness is d ifficu lt to
estimate but it has been reported that in 2005 alone around 76 million cases of food bo rne
diseases resu lting in 325000 hospita liza tions and 5000 dea ths are es timated to occur in the
United State (WHO 20 11 ) In addition food contaminat ion also causes enormous socia l and
economical burden on the community A consumer research repon released on 2010 revea led
that the healt h-related costs of food-borne illnesses had cost up to 152 billion do llar a year
includ ing the costs of med ica l bills lost wages and lost produc tivi ty (Smii middot 2010) Thus
food safety is cun-entl y an increasing important pub lic health issue laquougovenunents all over
the world are intensify ing the ir efforts to improve food safety
Among the majo r foodbome illness caused by mi croorgan ism are Salmonellosis
Campylobacteriosis Listeriosis and infectio ns due to entero haemonha gic (caJing intes tinal
bleeding) E coli (WHO 2007) III recent years Vibriosis had emerged to be an impOItant
bac te rial disease that canses human intestinal and ext ra-intestinal infections a ll over the world
(Chan elol 2002 Garc ia el al 2009 and Harth et al 2009) The three Vibrio species o f
primary significance in foodbo rne illnesses are V cholerae (serogroups 01 non-Ol and
2
- bull i
0 139) V vulnijiclIs and V paralwemolyricus Presently gast roenteitis caused by V
parahaemolyticus is extensively documented due to its increasing outbreaks (Alam el 01
2003 Gwendelynne el 01 2005 Gorny 2006 Harth et 01 2009 Roque et 01 2009)
V parahaemolyriclls is widely present in brackish and marine waters and infection to
human is regula rl y associated with the consumption of contaminated raw or undercooked
scafood (Guoxia ng el 01 2009) Ea rli er studies also demonstrated that V parahoemolylicus
can be easily iso lated from costal water sediments suspended particles and plankton due to
its halophi lic characteristic (Marlina el 01 2007 Zinuneman el 01 2007 Zu lkifli el al
2009 Vimala et 01 2010) However recent foodbome outbreaks throughout the world have
been intensive ly linked to consu mption of fresh fruits vegetables and unpasteurized juices
(Gorny 2006) Most recently Tunu ng et 01 (20 10) had rep0l1ed th at the vegetable samples
from supermarkets and wet markets in Selangor Malaysia were posit ive for pathogeni c
species of V parahaemolylicls Conversely there is no ot her known documented research on
the prevalence of this species in vegetables
In this st udy vegetables from two wet markets and two supem1arkets in Kuching
Sa rawak were sampled for the presence of V parallOemolyliclis The sa moles underwent
selective enrichment with alkaline peptone water (APW) supp lemented with 3 NaCI Then
presumptive V parahaemolYlicus were iso lated using TeES agar and CH ROMagar VibrioT
Following isolat ion gram stai ning together with a series of biochemica l tests were perfonned
to assist in the identification and confirmation of the iso lates
3
- bull i ~
IZ Objectives
This study was underta ken with the fo llowing objecti ves
I To isolate presumptive V parahaemolYlicus from marketed vegetabl es samples
in Kuching Sarawak by using Te BS and CHROMagar VibrioTM
II To confirm th e suspected V parahaemolyficus iso lates from marketed
vegetables samples in Kuching Sarawak by usi ng Gram stain and biochemical
tests
4
- bull i ~
USI KhJtlmal Makumat U umk VMVl~m ItLAYSlA AAAWAK
CHAPTER 2
LITERATURE REVIEW
21 The genus Vibrio
The genus Vibrio belongs to the fami ly Vibrionaceae and it consists of at least 34 recogn ized
species (HPA 2007a) In general Vibrio spp are Gram-negat ive fac ulta tively anaerobic
mOlile curved rods bacteria with a single polar flagellum (FEHD 2005) In surface waters of
the world Vibrios are among the mos t common surface organisms (Adeleye et aI 20 J 0)
They are present in both marine and fresh wa ter habiwts and commonly associa ted with
aquatic animals Vibrio pp such as V choerae V parahaemoylicus V vu nificus V
aginoylieus V mimicus V luvia lis V filmissii V melselmikovii V tollisae and V
dalltsea are common human pathogens (Tison 1999 Fi nkelstein 2000) T hey account for a
significant proportion of human infections as most of these vibrios secrete enterotoxins to
foods water or in the gastrointestinal tract (Nishibuchi and DePao la 2005)
22 Vibrio paralJlell1oytictls
Vibrio parahaemoy licus which is formerly known as Pasteurella parahaeliloytlca is a
non-sporing Gram-negat ive facu ltatively-anaerobic bacterium (Curti s el 01 2003) This
5
- ~
halophilic bacterium inhabits warm estuarine waters world wide (Bilung el al 2005) Thus il
reqlllres salt to survive and appears in high er concentrations during the warmer summer
months
The optimum temperature fo r the grow th of V parahaemolylicus is 37 degC and at Ihis
optimum temperature the bac terium has generation times of8 to 9 minutes in water and 12 [ 0
18 minutes in seafood (Montville and Matthews 2008) Thi s bacterium is the most IKeiy
Vibrio species to be imp licated in food borne diseases although both V vulnificus and V
cholera may also causes food bom e infections (Lawley e l al 2008) Clinical isolates of V
parrhaemolyticus produce beta type hemolysis on blood aga r (Wagatsuma agar) The
hemolysis is ca ll ed the Kanagawa-phenomenon (KP) which is associated with the production
ofa thermo stabl e d irect hemolys inTOH (Boyd 2008) TOH caused damage to the euka ryotic
cells by acting as a pore fonning toxin that alters the ion balance of cells (Boyd 2008)
Figure 21 SEM observa tlOos of V plrh cm)JJ(cu~
Image source Belas amp Colwe ll ( 1982)
6
-- UI
23 Fresh vegetables
Consumpti on of raw vegetables is common throughout the world not onl y owing to Ihe health
benefit but they are thought to be free from disease causing pathogens However recent food
borne outbreaks worldwi de is associ ated to consumption of fresh frui ts uncooked vegetables
and unpasteurized juices (Gorny 2006) Fruits and vegetable especially those that are eaten
raw without peeling can be the vehicles for transmission of a wide range of mi croorganis m
(Erdogru l and Sener 2005)
Based on Haeton and Jones (2008) E coli Salmonella spp and Listeria spp are
commonly present in vegetable samples Meldrum el 01 (2009) report ed that the two recent
large ou tbreaks in the United Kingdom showed signi fi cant health problems that cou ld ari se
from consumption of contaminated salad Another study of the preva lence of pathogens in
vegetables was reported by Little el 01 (2007) in which they found Listeria monocylOgenes in
ready-to-eat mixed salads in the United Kingdom Several other studi es on d ifferent
microorga nism isolated from vegetabl es were also report ed recent ly by local researchers
(Chai el 01 2008 Han el 01 2009 Ponni ah el 0 2009)
In Mala ys ia the presence of V parahoemo yticus has also been repOIt ed in raw
vegetables samples from wet markets and supeLl11arkets in Selangor (Tunung et 0 20 I 0)
This same study discovered that raw vegetabl es such as OenGnthe st l7i(era (Japanese
parsley) and C[cumis sali vus (Cucumber) were contaminated with virulent V
7
parahaemoiYlicus Consequen tly these vegetables could act as a transmission route and
poses risk to consumers after consumption Tunung el 01 (2011) had also demonstrated that
the vegetable samples fro m wet market contained higher levels of V paraiJaemolylicls as
compared w ith supennarkets The research suggested that there are many factors that can
contri bu te to the distribution of V parahaemoiylicus such as temperature holding ti me as
well as hygiene and sanitation practice (Tunung el aI 2011) However there is no known
documented research on V parahaemolYlicus in raw vegetables in Sarawak
24 Clinical characteristic of V parahaelllolyliclis infections
II parahaemoiyticus causes three distinct illness syndromes which are gastroenteritis wound
or soft tissues infections and septicemia (Sillljee 2007) The most freq uen t syndrome is
gastroeuteritis The symptoms include dianhea with abdomina l cramps nausea vo miting
headache and low-grade fever (S tarlin 2005 WilU1 and Konema n 2006 Montville and
Matthew 2008) Sometimes the diarrhea is bloody w ith stools described as meat washed
since tl1e stool is reddish and watery (Qadri el aI 2009)
As for wound or tis sue in fections this coudition occurs by the exposure of broken skin
or wound to warm seawater and seafood dripp ings (Ralph and Currie 2007 Simjee 2007)
In severe cases serious wound infection will resu lt in necrotizing fasc iiti s (Ralph and Curri e
2007) Although less common in V paraiwemolyliclIs infection septicemia is characteri7cd
by fever or hypotension with the isolation of V parahaemolyticus in the blood Septicemia
typicall y occurs in person with underlying health problems such as liver disease diabetes
cancer or other immune disorder (Simjee 2007) V parahaemoyticlis can even ca use fala l
septicemia in an immune-compromised hosts
The symptoms usuall y occur on the average of 15 hours but can began as early as 4
hours and as late as 36 hours after exposure and this will continue up to 3 days (Barceloux
200S) However these illnesses are usua lly categorized as a short illnesses and no prolonged
enect will take place after recovery (Wright et al 2009)
25 Isolation of V parahael1lolyliclIs
For the isolation of V parahaemoyticus an initial emichment using alka line peptone wat er
(APW) with pH S5 would be useful as this medium supports the growth uf viable ce lls
(Simjee 2007) Plati ng of emichment broth onto TCBS agar has been widely used for
selective isolation of V parahaemolytics from foods (Thompson el ai 2006 Oi Pinto et al
201 0 Canizalez -Roman et ai 2011 ) An average of 24 hours incubation at 37 degC is requ ired
fo r typica l V parahaemolylicus to develop colonies that are smooth gree n (s u~rose nega tive)
and 2 to 3 nun in di ameter on TCBS (ISO 2007) However V parahaenlOlytlcus co lonies on
TCBS agar are difficult to distinguish vi suall y from other bacterial colonies since they can be
covered by yellow color produced by sucrose-fennenting bacteria (Thompson el 01 2006
Rambach 2007)
9
_ ------~-- i ~~
Another widely used selective medium is CHROMagar VibrioT On this medium V
pllrahaemoiy liclis will developed mau ve color colonies (Kudo et aI 2001 Oi Pi nto 2010
Canizalez-Roman 2011) This agar medium contains substrates for beta-galactosidase which
was developed specifically to differentiate V parahaemolytics from ot her bacteria by us ing a
chromogenic su bstrate instead of sugar fermentation in traditional growth med ia such as
TCBS (Hara-Kudo et 01 2001) Therefore it is more sensitiv e and accurate for identi fying V
porahllemolyticlIs in food samples rather than the use of Te ES (Oi Pinto 2010 Canizalez-
Roman 20 II)
26 Identification through Gram-stain and biochemical tests
V parahaemoiyticus is a Gram-negative bacterium th us this organi sm stained pink or red
after Gram staining (HPA 20073) Under microscopic observation a typical V
pllrahaemolyticus appeared as rod characterist ica lly curved or comma-shoped The bacterium
is also positi ve for motility under microscopic observations (ISO 2007 BPA 2007a)
V parahaemolylieus ca n be identified through a seri es of biochemical tests According
to Khan et 01 (2007) V para haemolyticus can be distinct ively differentia ted fro m the other
Vibrio spp by performing sa lt tolerance and acid fro m femlen tation tests A typical V
parahaemoiYlicus co lonies would not grow in AFW without th e presence of at least 3 of sali
10
~-------
but it can only endure the presence of salt up to 8 (ISO 2007 Khan el aI 2007) In the
other hand the fermentation test for acid production is the most important test to differentiate
between V parahaemolyliclIs and V vulnificus species (Khan el aI 2007) Jayasinge et ai
(2010) even suggested that the combination of salt toleranc e test Voges-Proskauer test and
ONPG test can provide a strai ghtforward differentiation of V parahaemoly liclis from the
other Vibrionaceae species (Figure 31)
Citrate test screens the ab ilit y of bacterial isolates to utilize ci trate as its carbon and
energy source (MacFaddin 2000) This test is a very important test to identify gram-negative
pathogens (Tang el aI 1998) The MR-VP medium which was used for Met hyl red and
Voges Proskauer test contains glucose and peptone Methyl red test screens for the bacterial
metaboli sm pathways for glucose fermentation while the Voges-Proskauer tes t is an assay for
organisms which ferment glucose with only one fermentation product usuall y acetic acid
The TS[ test is another useful test tha t can distinguish V parahaemolyticus from other
bacteria (ISO 2007) The TSJ agar med ium contains peptone fenous sulfate lactose sucrose
and a small amount of glucose (dex trose) The pH indi cator is phenol red This medium
differentiates bacteria based on their ability to fennent glucose lactose ando r sucrose and on
their abi lity to reduce sulfur to hydrogen sulfide (H S) The T SJ test also haj the abi lity to
determine the production of H]S (Kim et aI 1999)
In additi on other bi ochemica l tests such as oxidase lysine decarboxylase om ithi ne
decarboxylase ca talase and indole can also be used for the purpose of identifying V
II
-- ---- - ----------- - ~i
parahaemoyticus iso lates (ISO 2007 Jayasinghe et ai 2007 Khan et aI 2007) The
biochemical reactions o f V parahaemoiyticus are summarized in Table 21
Table 21 Siandard biochemical tesl reactions of V porahaemoyticus
TeSi S Reaction of V parahaem olyaclIs Refe rences Salt tol ance
+
Sali ne TSI agar KIA
No HS and gas production
Simmons Citrnh
Voges-Proskauer
Methyl-Red +
[SO (2007)
Jayasinge et al (2007)
Khan et of (2007)
ISO (2007)
Kim et I (1999)
Abide et til (1999)
Kim 11111 (1999)
Khan et til (2007)
Jayasinge el III (20 (0)
Kim et 0 (2 007)
Note (-) ~ nega live (+) ~ positi ve K ~ alka line ~ aC idic
12
CHAPT ER 3
MATERIALS AND METI IODS
31 Samples collection
Eighty raw vegetable samples were collected week ly from two wet markets (WI amp W2) and
two supennarkets (S 1 amp S2) in Kuching Sarawa k The collected samples consisted o f four
di fferent groups of vegetables which are regularly consumed ra w by the locals These samples
wcr~ rand omly selected within ten weeks -period which is from October 20 10 to January
20 11 Duri ng collection each sample was transferred to clean plastic bags labeled acco rding
to the place they were collected and were analyzed immed iately on arriva l to the labo rat ory
The types of raw vegetable sa mples obtained were as shown in Table 31
Tab le 31 Types o f raw vegetable samples co llected from wet markes and supcmlarkels
Group Loca l nam e English nam e Scienti fi c Home l mple coding No or samples
Soil Kubis Cabbage Brassica v eracea K 20 Roots Lobak Canmiddotot Douclfs carola C ~O
mera h Beans KaCtlO g Four-winged PsophOCaJjJ lls KB cO
bow l bean JeIlagulIolobus Shoot Tauge Bean-I_prout Vigna rad mc T 10 Total 80
13
---------~ I I
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
ACKNOWLpoundDCE~1ENT
Thanks be to God His grace and blessings had guided me to the completi on of thi s fin al year
project and a ll along my 3 years of studying in UNIMAS He is whom ltumed to in times of
weary and confusion By His will this project was finall y completed
First and forem ost I would li ke to express my deepest gra titud e and appreciations to
my respec ted supervisor Dr Lesley Maurice Bi lung for her endless eff0l1 advice suggesti on
and supervision I am also very grateful to have Dr Micky Vincent as my co -supervisor who
has been so helpfu l Not forge tting thanks a lott o Dr Samu el Lihan who assis ted in the fin al
editing of this writing
My gratefulness is a lso extended to a ll the postgraduate students in Microb iology Jab
especially Vel nett i and Ka thl een for their countless help They have been so generous in
sharing their know ledge and never fa iled to lend a helping hand in my times of d ifficulties
This project wouldn t have been a success wi tho ut the both of them tha nks and all the best
for the future l My thankfulness also reached ou t to all m y lab mates Mim Ard i Lazarus
Sheila Tata ng Matthew and the others for being such wonderful considerate and help[d
fr iends My hea11felt apprec ia tion also goes to my lovely parents sib lings fri ends and
beloved ones fo r the ir unfai ling love cons istent prayers and constant suppol1 Last but not
least thank you to a ll my lecturers course mates nnd everyone that has con tributed eit her
direc tly or ind irectl y tow ards the completion of this project
)
-~-- l
DECLARATION
I hereby declare thnt this Fina l Year Project report is based on m y origina l work except for
quotations and citations which have been properly acknowledged [ also declare that it has not
been previously or concurrentl y subm illed fo r any other degree in UNIMAS or other in stitut ions
SABATINI JU- OB (222 18)
Date 305 gt01
11
~
bullbull
TABLE OF CONTENTS
ACKN OWLEI)GEMENT
DECLARATJO II
TABLE OF CONTENTS I II
LIST OF ABBREVIATIONS v
LIST OF TABLES D FIGURES V I
ABSTRACT I ABSTRAK
CHAPTER 1
CHAPTER 2
CH PTER3
INTRODUCTION 2
Lrt ERATlJRE REVIEWS 5
21 The genus Vibrio 5
22 Vibrio parahaemolylicus 5
23 Fresh vegetables 7
24 Clinica l cha ract eristics of V parahaemolyticus 8
infections
25 Iso lation of V parahaemolyticus 9
26 Id entilication through Gra m-Stain and biochemical 10
tes ts
JllATERlAL AND METHODS 13
31 Samples co ll ection 13
32 Samples preparat ion and enrichment 14
III
__a ------ _bull 1 shy
33 Isolation o f V parahaemolylicus 14
34 Preparation for Gram-stain and biochemical tests 15
35 G ram slain prolocol 16
36 Biochemicallesls 16
36 1 Voges-Proskauer test 18
362 Methyl-Red test 18
363 Sa lt tolerance test IS
36 4 Triple sugar iron test (TSI) 19
365 Hyd rogen sulfid e and gas production 19
366 Ci trate test 19
RESULTS 20
41 Iso lation of V parahaemolyticlIs 20
43 Gram-stai n and biochemical tests 23
CHAPTER 5 DI CUSSION 28
51 Vegetab les types 28
52 Co loni es appearance on selective aga r 28
53 Isolation rate of V paraiaemolylicus on TCa and 30
C HROMagar Vibrio
54 In terpre ta tions of biochemical tests 34
55 Preva lence of V parahaemolylicus 35
CIIAPTER 6 CONCLUS ION 37
CHAPTER 7 REFERENCES 39
IV
-shy
--
C
APW
g
KJA
NK
1111
mm
MR
NaCI
PCR
Spp
TeBS
TSI
VP
V parahaemoLytiCLIs Vp
WHO
LIST OF ABBREVIATIONS
percentage
degree celcius
Alkaline Peptone Water
gram
hyd rogen sulfite
alkaline slant acidic bUlt
acidic slant alkaline butt
microliter
milli liter
milimeter
Methyl-red
Sodium chloride
Polymerase Chain Reaction
species
Tiosulphate Citrate Bile Salt Sucrose
Tliple Sugar Iron
V oges-Proskaeur
Vibrio parallOemolyticlIs
World Heal th Organizati ons
v
~-
LIST OF TABLES
Tables Page
Table 21 Standard biochemical test reactio ns of V parahaemolyliclIs 12
Table 31 Types of raw vegetable samples collected from wet markets and 13
supermarkets
Table 41 Results for the presence of V parahaemolyticus on TCBS and 2 1
CHROMa gar Vibrio from the vegetable sa mples
Table 42 Result s of microscopic observations of the suspected V 27
parahaemolylicus iso lates
Table 43 Overall resuit of biochemical tests 24
vi
--bull i~
LIST OF FIG URES
Figures Page
Figurc 2t SEM observation o f Ii parahaemolyticus Image source Belas amp 6
Colwell (1 982)
and C I-IR OM agar Vib rio trade based on different sam pling locr io ns
Figure 31 Biochemical keys fo r the identification o f Vibrio species 17
Figure 41 The isolation o f Ii parQhaemolyticus on TCBS 22
Figure 42 Appearance of mixed colonies on CHROM agar Vib riotrade 22
Figure 43 Pure mauve co lonies obtained a fter sub -culturing 23
figurc 44 Reac tio ns of (a) B7KBS and (b) B8CW towards Voges -Proskauer test 24
Figure 45 Reactio ns of (a) B7KBS and (b) BI CW towards M dhly-red test 25
Figure 46 The reaction of a ll isolates towards citrate test 25
Figure 47 The reaction o f (a) B7KBS = KJA and (b) B3KW = AK fo r TSltes t 26
Figure 48 The reactio n o fB7KBS towards (a) 0 NaC I and (b) 8 Nae 27
Figu re 5 1 Bar graph o f the di stribution of presumptive pas iti ve sampl es on TCBS 30
Figure 52 Pie chart o f the distribu tion of presumptive Ii paraliaemolyliclls 31
between different vegetable types on CHROM aga r Vibrio
Figure 53 Pi e chart of the distribution of presumptive Ii parahaemolylicus 33
between di fferent vegetab le types on CHROMagar Vibriotrade
VII
--~bull i lj
Tbe Incidence of Vibrio p arahaellloly ticlis in Raw Vegetables at Local Wet Markets and Supermarkets in Kuching Sarawak
Sabatini lihob
Resource BIOIClhnoltlg) Progrtlm me Facu Ity or Resource Science Bud T~lln(llogy
Uni versity Malaysb Sa rawa k
ABSTRACT
Vibrio parawemoyliCLls is a foodbome pa thogen that is regular ly assoc iated with the consu mption o f
raw or undercooked seafood The mos t frequent sympto m of V parahaemoylicus infecr ion is
gastroenteritis Recen tly this pa thogen was also disco vered in raw vegetable samples in Peninsular
Malaysia Thus the aim of this study is to isolate and de tect V paahaemoylicu~ in raw vegdables T he samples which consisted of carrot fourmiddot winged bean cabbage and bea n sp ro ul were purc hased
tiom two wet markets and two supenn arkets in Kucll ing Sarawak In th is stud y alkaline peplone
water (A PW) wa s used as the enridunent medium The combinati o n o f two types o f selective agar
TCBS and CHROMa gar Vibrio nbull1 was successfully app lied to isolate the bacteria A fte r a series of
biochemical tests only 2 out of the 80 samples were pos itive for the p resence of V parahaemoylicLls
Although the peroentage of V pOrehaemolylics prevalence was low (2 5) the resu lts h ighli ghted
the fac t that raw vege tables could also be contaminated w It h V parahaenro~Vlicus
Key words Vib rio parahemotyfic~ raw vegetables TC BS and CHROMagar Vibrio rM
ABSTRAK
Vibrio parawcmoylicus merupakan sejenis patogen bmvaan makanafl )long sering dikairkan dengan
pengambiall makonon laul seewa men fa h Tonda paling kemp bagi j angkitall k parahwnJ r lvticlfs
ialah gasroenlritis TerbolU pafogen ini Ina dijumpa i di dalam sUlllp~1 scJyuran melJfah di
Semenanjung A1nlaYiia Kajial1 ini berlujuan un uk mengasmg dan mengesan V nanlhaemolliqi di
daall1 sampel $oyuran menfah Sampel yang lerdiri doripada obok merah kac(mg botol kobis dan
(l uge teah dlbeli dari dU(J pasai basah Clnd clua pasar raya di Kuchi lg Sorm lak Il l pepton beralktrli
(A PW) fel(lh digul1akan Ill1tuk memperbonyakko1 jwn Jah bakfcna Kombilw si pelggunaon agar
terpilih Tess dan CHROkiagar Vibrio T1 tdoh bejaya mel11ellcilkon ba( ur ia yallg dikehendoki
Selelah beberapa siri lljion biokimia didapafi 2 daripada 80 sampel relah dlcemQj oleh 1 rar(fhoenlJliJji(u~ Mcsklpun perafus kehadiran V norahaemolvticus adalah remJah (25~ o)
kepuflfsan kajian ini menunjukkan bahawo saYllran mental boleh dicemari ole Y paraha~moh IIcm
Kata klllt ci Vibrio oorqhaemol liclfs sayuran men fah TCBS dOll CHROtviagar Vihriotrade
-
CHAPTER 1
INTRODUCTION
11 Introduction
Foodbome illness assoc iated with consumption of contam inated food is a corrunon hea lth
problem that occurs worldwide The global inc idence of food bo rne ill ness is d ifficu lt to
estimate but it has been reported that in 2005 alone around 76 million cases of food bo rne
diseases resu lting in 325000 hospita liza tions and 5000 dea ths are es timated to occur in the
United State (WHO 20 11 ) In addition food contaminat ion also causes enormous socia l and
economical burden on the community A consumer research repon released on 2010 revea led
that the healt h-related costs of food-borne illnesses had cost up to 152 billion do llar a year
includ ing the costs of med ica l bills lost wages and lost produc tivi ty (Smii middot 2010) Thus
food safety is cun-entl y an increasing important pub lic health issue laquougovenunents all over
the world are intensify ing the ir efforts to improve food safety
Among the majo r foodbome illness caused by mi croorgan ism are Salmonellosis
Campylobacteriosis Listeriosis and infectio ns due to entero haemonha gic (caJing intes tinal
bleeding) E coli (WHO 2007) III recent years Vibriosis had emerged to be an impOItant
bac te rial disease that canses human intestinal and ext ra-intestinal infections a ll over the world
(Chan elol 2002 Garc ia el al 2009 and Harth et al 2009) The three Vibrio species o f
primary significance in foodbo rne illnesses are V cholerae (serogroups 01 non-Ol and
2
- bull i
0 139) V vulnijiclIs and V paralwemolyricus Presently gast roenteitis caused by V
parahaemolyticus is extensively documented due to its increasing outbreaks (Alam el 01
2003 Gwendelynne el 01 2005 Gorny 2006 Harth et 01 2009 Roque et 01 2009)
V parahaemolyriclls is widely present in brackish and marine waters and infection to
human is regula rl y associated with the consumption of contaminated raw or undercooked
scafood (Guoxia ng el 01 2009) Ea rli er studies also demonstrated that V parahoemolylicus
can be easily iso lated from costal water sediments suspended particles and plankton due to
its halophi lic characteristic (Marlina el 01 2007 Zinuneman el 01 2007 Zu lkifli el al
2009 Vimala et 01 2010) However recent foodbome outbreaks throughout the world have
been intensive ly linked to consu mption of fresh fruits vegetables and unpasteurized juices
(Gorny 2006) Most recently Tunu ng et 01 (20 10) had rep0l1ed th at the vegetable samples
from supermarkets and wet markets in Selangor Malaysia were posit ive for pathogeni c
species of V parahaemolylicls Conversely there is no ot her known documented research on
the prevalence of this species in vegetables
In this st udy vegetables from two wet markets and two supem1arkets in Kuching
Sa rawak were sampled for the presence of V parallOemolyliclis The sa moles underwent
selective enrichment with alkaline peptone water (APW) supp lemented with 3 NaCI Then
presumptive V parahaemolYlicus were iso lated using TeES agar and CH ROMagar VibrioT
Following isolat ion gram stai ning together with a series of biochemica l tests were perfonned
to assist in the identification and confirmation of the iso lates
3
- bull i ~
IZ Objectives
This study was underta ken with the fo llowing objecti ves
I To isolate presumptive V parahaemolYlicus from marketed vegetabl es samples
in Kuching Sarawak by using Te BS and CHROMagar VibrioTM
II To confirm th e suspected V parahaemolyficus iso lates from marketed
vegetables samples in Kuching Sarawak by usi ng Gram stain and biochemical
tests
4
- bull i ~
USI KhJtlmal Makumat U umk VMVl~m ItLAYSlA AAAWAK
CHAPTER 2
LITERATURE REVIEW
21 The genus Vibrio
The genus Vibrio belongs to the fami ly Vibrionaceae and it consists of at least 34 recogn ized
species (HPA 2007a) In general Vibrio spp are Gram-negat ive fac ulta tively anaerobic
mOlile curved rods bacteria with a single polar flagellum (FEHD 2005) In surface waters of
the world Vibrios are among the mos t common surface organisms (Adeleye et aI 20 J 0)
They are present in both marine and fresh wa ter habiwts and commonly associa ted with
aquatic animals Vibrio pp such as V choerae V parahaemoylicus V vu nificus V
aginoylieus V mimicus V luvia lis V filmissii V melselmikovii V tollisae and V
dalltsea are common human pathogens (Tison 1999 Fi nkelstein 2000) T hey account for a
significant proportion of human infections as most of these vibrios secrete enterotoxins to
foods water or in the gastrointestinal tract (Nishibuchi and DePao la 2005)
22 Vibrio paralJlell1oytictls
Vibrio parahaemoy licus which is formerly known as Pasteurella parahaeliloytlca is a
non-sporing Gram-negat ive facu ltatively-anaerobic bacterium (Curti s el 01 2003) This
5
- ~
halophilic bacterium inhabits warm estuarine waters world wide (Bilung el al 2005) Thus il
reqlllres salt to survive and appears in high er concentrations during the warmer summer
months
The optimum temperature fo r the grow th of V parahaemolylicus is 37 degC and at Ihis
optimum temperature the bac terium has generation times of8 to 9 minutes in water and 12 [ 0
18 minutes in seafood (Montville and Matthews 2008) Thi s bacterium is the most IKeiy
Vibrio species to be imp licated in food borne diseases although both V vulnificus and V
cholera may also causes food bom e infections (Lawley e l al 2008) Clinical isolates of V
parrhaemolyticus produce beta type hemolysis on blood aga r (Wagatsuma agar) The
hemolysis is ca ll ed the Kanagawa-phenomenon (KP) which is associated with the production
ofa thermo stabl e d irect hemolys inTOH (Boyd 2008) TOH caused damage to the euka ryotic
cells by acting as a pore fonning toxin that alters the ion balance of cells (Boyd 2008)
Figure 21 SEM observa tlOos of V plrh cm)JJ(cu~
Image source Belas amp Colwe ll ( 1982)
6
-- UI
23 Fresh vegetables
Consumpti on of raw vegetables is common throughout the world not onl y owing to Ihe health
benefit but they are thought to be free from disease causing pathogens However recent food
borne outbreaks worldwi de is associ ated to consumption of fresh frui ts uncooked vegetables
and unpasteurized juices (Gorny 2006) Fruits and vegetable especially those that are eaten
raw without peeling can be the vehicles for transmission of a wide range of mi croorganis m
(Erdogru l and Sener 2005)
Based on Haeton and Jones (2008) E coli Salmonella spp and Listeria spp are
commonly present in vegetable samples Meldrum el 01 (2009) report ed that the two recent
large ou tbreaks in the United Kingdom showed signi fi cant health problems that cou ld ari se
from consumption of contaminated salad Another study of the preva lence of pathogens in
vegetables was reported by Little el 01 (2007) in which they found Listeria monocylOgenes in
ready-to-eat mixed salads in the United Kingdom Several other studi es on d ifferent
microorga nism isolated from vegetabl es were also report ed recent ly by local researchers
(Chai el 01 2008 Han el 01 2009 Ponni ah el 0 2009)
In Mala ys ia the presence of V parahoemo yticus has also been repOIt ed in raw
vegetables samples from wet markets and supeLl11arkets in Selangor (Tunung et 0 20 I 0)
This same study discovered that raw vegetabl es such as OenGnthe st l7i(era (Japanese
parsley) and C[cumis sali vus (Cucumber) were contaminated with virulent V
7
parahaemoiYlicus Consequen tly these vegetables could act as a transmission route and
poses risk to consumers after consumption Tunung el 01 (2011) had also demonstrated that
the vegetable samples fro m wet market contained higher levels of V paraiJaemolylicls as
compared w ith supennarkets The research suggested that there are many factors that can
contri bu te to the distribution of V parahaemoiylicus such as temperature holding ti me as
well as hygiene and sanitation practice (Tunung el aI 2011) However there is no known
documented research on V parahaemolYlicus in raw vegetables in Sarawak
24 Clinical characteristic of V parahaelllolyliclis infections
II parahaemoiyticus causes three distinct illness syndromes which are gastroenteritis wound
or soft tissues infections and septicemia (Sillljee 2007) The most freq uen t syndrome is
gastroeuteritis The symptoms include dianhea with abdomina l cramps nausea vo miting
headache and low-grade fever (S tarlin 2005 WilU1 and Konema n 2006 Montville and
Matthew 2008) Sometimes the diarrhea is bloody w ith stools described as meat washed
since tl1e stool is reddish and watery (Qadri el aI 2009)
As for wound or tis sue in fections this coudition occurs by the exposure of broken skin
or wound to warm seawater and seafood dripp ings (Ralph and Currie 2007 Simjee 2007)
In severe cases serious wound infection will resu lt in necrotizing fasc iiti s (Ralph and Curri e
2007) Although less common in V paraiwemolyliclIs infection septicemia is characteri7cd
by fever or hypotension with the isolation of V parahaemolyticus in the blood Septicemia
typicall y occurs in person with underlying health problems such as liver disease diabetes
cancer or other immune disorder (Simjee 2007) V parahaemoyticlis can even ca use fala l
septicemia in an immune-compromised hosts
The symptoms usuall y occur on the average of 15 hours but can began as early as 4
hours and as late as 36 hours after exposure and this will continue up to 3 days (Barceloux
200S) However these illnesses are usua lly categorized as a short illnesses and no prolonged
enect will take place after recovery (Wright et al 2009)
25 Isolation of V parahael1lolyliclIs
For the isolation of V parahaemoyticus an initial emichment using alka line peptone wat er
(APW) with pH S5 would be useful as this medium supports the growth uf viable ce lls
(Simjee 2007) Plati ng of emichment broth onto TCBS agar has been widely used for
selective isolation of V parahaemolytics from foods (Thompson el ai 2006 Oi Pinto et al
201 0 Canizalez -Roman et ai 2011 ) An average of 24 hours incubation at 37 degC is requ ired
fo r typica l V parahaemolylicus to develop colonies that are smooth gree n (s u~rose nega tive)
and 2 to 3 nun in di ameter on TCBS (ISO 2007) However V parahaenlOlytlcus co lonies on
TCBS agar are difficult to distinguish vi suall y from other bacterial colonies since they can be
covered by yellow color produced by sucrose-fennenting bacteria (Thompson el 01 2006
Rambach 2007)
9
_ ------~-- i ~~
Another widely used selective medium is CHROMagar VibrioT On this medium V
pllrahaemoiy liclis will developed mau ve color colonies (Kudo et aI 2001 Oi Pi nto 2010
Canizalez-Roman 2011) This agar medium contains substrates for beta-galactosidase which
was developed specifically to differentiate V parahaemolytics from ot her bacteria by us ing a
chromogenic su bstrate instead of sugar fermentation in traditional growth med ia such as
TCBS (Hara-Kudo et 01 2001) Therefore it is more sensitiv e and accurate for identi fying V
porahllemolyticlIs in food samples rather than the use of Te ES (Oi Pinto 2010 Canizalez-
Roman 20 II)
26 Identification through Gram-stain and biochemical tests
V parahaemoiyticus is a Gram-negative bacterium th us this organi sm stained pink or red
after Gram staining (HPA 20073) Under microscopic observation a typical V
pllrahaemolyticus appeared as rod characterist ica lly curved or comma-shoped The bacterium
is also positi ve for motility under microscopic observations (ISO 2007 BPA 2007a)
V parahaemolylieus ca n be identified through a seri es of biochemical tests According
to Khan et 01 (2007) V para haemolyticus can be distinct ively differentia ted fro m the other
Vibrio spp by performing sa lt tolerance and acid fro m femlen tation tests A typical V
parahaemoiYlicus co lonies would not grow in AFW without th e presence of at least 3 of sali
10
~-------
but it can only endure the presence of salt up to 8 (ISO 2007 Khan el aI 2007) In the
other hand the fermentation test for acid production is the most important test to differentiate
between V parahaemolyliclIs and V vulnificus species (Khan el aI 2007) Jayasinge et ai
(2010) even suggested that the combination of salt toleranc e test Voges-Proskauer test and
ONPG test can provide a strai ghtforward differentiation of V parahaemoly liclis from the
other Vibrionaceae species (Figure 31)
Citrate test screens the ab ilit y of bacterial isolates to utilize ci trate as its carbon and
energy source (MacFaddin 2000) This test is a very important test to identify gram-negative
pathogens (Tang el aI 1998) The MR-VP medium which was used for Met hyl red and
Voges Proskauer test contains glucose and peptone Methyl red test screens for the bacterial
metaboli sm pathways for glucose fermentation while the Voges-Proskauer tes t is an assay for
organisms which ferment glucose with only one fermentation product usuall y acetic acid
The TS[ test is another useful test tha t can distinguish V parahaemolyticus from other
bacteria (ISO 2007) The TSJ agar med ium contains peptone fenous sulfate lactose sucrose
and a small amount of glucose (dex trose) The pH indi cator is phenol red This medium
differentiates bacteria based on their ability to fennent glucose lactose ando r sucrose and on
their abi lity to reduce sulfur to hydrogen sulfide (H S) The T SJ test also haj the abi lity to
determine the production of H]S (Kim et aI 1999)
In additi on other bi ochemica l tests such as oxidase lysine decarboxylase om ithi ne
decarboxylase ca talase and indole can also be used for the purpose of identifying V
II
-- ---- - ----------- - ~i
parahaemoyticus iso lates (ISO 2007 Jayasinghe et ai 2007 Khan et aI 2007) The
biochemical reactions o f V parahaemoiyticus are summarized in Table 21
Table 21 Siandard biochemical tesl reactions of V porahaemoyticus
TeSi S Reaction of V parahaem olyaclIs Refe rences Salt tol ance
+
Sali ne TSI agar KIA
No HS and gas production
Simmons Citrnh
Voges-Proskauer
Methyl-Red +
[SO (2007)
Jayasinge et al (2007)
Khan et of (2007)
ISO (2007)
Kim et I (1999)
Abide et til (1999)
Kim 11111 (1999)
Khan et til (2007)
Jayasinge el III (20 (0)
Kim et 0 (2 007)
Note (-) ~ nega live (+) ~ positi ve K ~ alka line ~ aC idic
12
CHAPT ER 3
MATERIALS AND METI IODS
31 Samples collection
Eighty raw vegetable samples were collected week ly from two wet markets (WI amp W2) and
two supennarkets (S 1 amp S2) in Kuching Sarawa k The collected samples consisted o f four
di fferent groups of vegetables which are regularly consumed ra w by the locals These samples
wcr~ rand omly selected within ten weeks -period which is from October 20 10 to January
20 11 Duri ng collection each sample was transferred to clean plastic bags labeled acco rding
to the place they were collected and were analyzed immed iately on arriva l to the labo rat ory
The types of raw vegetable sa mples obtained were as shown in Table 31
Tab le 31 Types o f raw vegetable samples co llected from wet markes and supcmlarkels
Group Loca l nam e English nam e Scienti fi c Home l mple coding No or samples
Soil Kubis Cabbage Brassica v eracea K 20 Roots Lobak Canmiddotot Douclfs carola C ~O
mera h Beans KaCtlO g Four-winged PsophOCaJjJ lls KB cO
bow l bean JeIlagulIolobus Shoot Tauge Bean-I_prout Vigna rad mc T 10 Total 80
13
---------~ I I
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
DECLARATION
I hereby declare thnt this Fina l Year Project report is based on m y origina l work except for
quotations and citations which have been properly acknowledged [ also declare that it has not
been previously or concurrentl y subm illed fo r any other degree in UNIMAS or other in stitut ions
SABATINI JU- OB (222 18)
Date 305 gt01
11
~
bullbull
TABLE OF CONTENTS
ACKN OWLEI)GEMENT
DECLARATJO II
TABLE OF CONTENTS I II
LIST OF ABBREVIATIONS v
LIST OF TABLES D FIGURES V I
ABSTRACT I ABSTRAK
CHAPTER 1
CHAPTER 2
CH PTER3
INTRODUCTION 2
Lrt ERATlJRE REVIEWS 5
21 The genus Vibrio 5
22 Vibrio parahaemolylicus 5
23 Fresh vegetables 7
24 Clinica l cha ract eristics of V parahaemolyticus 8
infections
25 Iso lation of V parahaemolyticus 9
26 Id entilication through Gra m-Stain and biochemical 10
tes ts
JllATERlAL AND METHODS 13
31 Samples co ll ection 13
32 Samples preparat ion and enrichment 14
III
__a ------ _bull 1 shy
33 Isolation o f V parahaemolylicus 14
34 Preparation for Gram-stain and biochemical tests 15
35 G ram slain prolocol 16
36 Biochemicallesls 16
36 1 Voges-Proskauer test 18
362 Methyl-Red test 18
363 Sa lt tolerance test IS
36 4 Triple sugar iron test (TSI) 19
365 Hyd rogen sulfid e and gas production 19
366 Ci trate test 19
RESULTS 20
41 Iso lation of V parahaemolyticlIs 20
43 Gram-stai n and biochemical tests 23
CHAPTER 5 DI CUSSION 28
51 Vegetab les types 28
52 Co loni es appearance on selective aga r 28
53 Isolation rate of V paraiaemolylicus on TCa and 30
C HROMagar Vibrio
54 In terpre ta tions of biochemical tests 34
55 Preva lence of V parahaemolylicus 35
CIIAPTER 6 CONCLUS ION 37
CHAPTER 7 REFERENCES 39
IV
-shy
--
C
APW
g
KJA
NK
1111
mm
MR
NaCI
PCR
Spp
TeBS
TSI
VP
V parahaemoLytiCLIs Vp
WHO
LIST OF ABBREVIATIONS
percentage
degree celcius
Alkaline Peptone Water
gram
hyd rogen sulfite
alkaline slant acidic bUlt
acidic slant alkaline butt
microliter
milli liter
milimeter
Methyl-red
Sodium chloride
Polymerase Chain Reaction
species
Tiosulphate Citrate Bile Salt Sucrose
Tliple Sugar Iron
V oges-Proskaeur
Vibrio parallOemolyticlIs
World Heal th Organizati ons
v
~-
LIST OF TABLES
Tables Page
Table 21 Standard biochemical test reactio ns of V parahaemolyliclIs 12
Table 31 Types of raw vegetable samples collected from wet markets and 13
supermarkets
Table 41 Results for the presence of V parahaemolyticus on TCBS and 2 1
CHROMa gar Vibrio from the vegetable sa mples
Table 42 Result s of microscopic observations of the suspected V 27
parahaemolylicus iso lates
Table 43 Overall resuit of biochemical tests 24
vi
--bull i~
LIST OF FIG URES
Figures Page
Figurc 2t SEM observation o f Ii parahaemolyticus Image source Belas amp 6
Colwell (1 982)
and C I-IR OM agar Vib rio trade based on different sam pling locr io ns
Figure 31 Biochemical keys fo r the identification o f Vibrio species 17
Figure 41 The isolation o f Ii parQhaemolyticus on TCBS 22
Figure 42 Appearance of mixed colonies on CHROM agar Vib riotrade 22
Figure 43 Pure mauve co lonies obtained a fter sub -culturing 23
figurc 44 Reac tio ns of (a) B7KBS and (b) B8CW towards Voges -Proskauer test 24
Figure 45 Reactio ns of (a) B7KBS and (b) BI CW towards M dhly-red test 25
Figure 46 The reaction of a ll isolates towards citrate test 25
Figure 47 The reaction o f (a) B7KBS = KJA and (b) B3KW = AK fo r TSltes t 26
Figure 48 The reactio n o fB7KBS towards (a) 0 NaC I and (b) 8 Nae 27
Figu re 5 1 Bar graph o f the di stribution of presumptive pas iti ve sampl es on TCBS 30
Figure 52 Pie chart o f the distribu tion of presumptive Ii paraliaemolyliclls 31
between different vegetable types on CHROM aga r Vibrio
Figure 53 Pi e chart of the distribution of presumptive Ii parahaemolylicus 33
between di fferent vegetab le types on CHROMagar Vibriotrade
VII
--~bull i lj
Tbe Incidence of Vibrio p arahaellloly ticlis in Raw Vegetables at Local Wet Markets and Supermarkets in Kuching Sarawak
Sabatini lihob
Resource BIOIClhnoltlg) Progrtlm me Facu Ity or Resource Science Bud T~lln(llogy
Uni versity Malaysb Sa rawa k
ABSTRACT
Vibrio parawemoyliCLls is a foodbome pa thogen that is regular ly assoc iated with the consu mption o f
raw or undercooked seafood The mos t frequent sympto m of V parahaemoylicus infecr ion is
gastroenteritis Recen tly this pa thogen was also disco vered in raw vegetable samples in Peninsular
Malaysia Thus the aim of this study is to isolate and de tect V paahaemoylicu~ in raw vegdables T he samples which consisted of carrot fourmiddot winged bean cabbage and bea n sp ro ul were purc hased
tiom two wet markets and two supenn arkets in Kucll ing Sarawak In th is stud y alkaline peplone
water (A PW) wa s used as the enridunent medium The combinati o n o f two types o f selective agar
TCBS and CHROMa gar Vibrio nbull1 was successfully app lied to isolate the bacteria A fte r a series of
biochemical tests only 2 out of the 80 samples were pos itive for the p resence of V parahaemoylicLls
Although the peroentage of V pOrehaemolylics prevalence was low (2 5) the resu lts h ighli ghted
the fac t that raw vege tables could also be contaminated w It h V parahaenro~Vlicus
Key words Vib rio parahemotyfic~ raw vegetables TC BS and CHROMagar Vibrio rM
ABSTRAK
Vibrio parawcmoylicus merupakan sejenis patogen bmvaan makanafl )long sering dikairkan dengan
pengambiall makonon laul seewa men fa h Tonda paling kemp bagi j angkitall k parahwnJ r lvticlfs
ialah gasroenlritis TerbolU pafogen ini Ina dijumpa i di dalam sUlllp~1 scJyuran melJfah di
Semenanjung A1nlaYiia Kajial1 ini berlujuan un uk mengasmg dan mengesan V nanlhaemolliqi di
daall1 sampel $oyuran menfah Sampel yang lerdiri doripada obok merah kac(mg botol kobis dan
(l uge teah dlbeli dari dU(J pasai basah Clnd clua pasar raya di Kuchi lg Sorm lak Il l pepton beralktrli
(A PW) fel(lh digul1akan Ill1tuk memperbonyakko1 jwn Jah bakfcna Kombilw si pelggunaon agar
terpilih Tess dan CHROkiagar Vibrio T1 tdoh bejaya mel11ellcilkon ba( ur ia yallg dikehendoki
Selelah beberapa siri lljion biokimia didapafi 2 daripada 80 sampel relah dlcemQj oleh 1 rar(fhoenlJliJji(u~ Mcsklpun perafus kehadiran V norahaemolvticus adalah remJah (25~ o)
kepuflfsan kajian ini menunjukkan bahawo saYllran mental boleh dicemari ole Y paraha~moh IIcm
Kata klllt ci Vibrio oorqhaemol liclfs sayuran men fah TCBS dOll CHROtviagar Vihriotrade
-
CHAPTER 1
INTRODUCTION
11 Introduction
Foodbome illness assoc iated with consumption of contam inated food is a corrunon hea lth
problem that occurs worldwide The global inc idence of food bo rne ill ness is d ifficu lt to
estimate but it has been reported that in 2005 alone around 76 million cases of food bo rne
diseases resu lting in 325000 hospita liza tions and 5000 dea ths are es timated to occur in the
United State (WHO 20 11 ) In addition food contaminat ion also causes enormous socia l and
economical burden on the community A consumer research repon released on 2010 revea led
that the healt h-related costs of food-borne illnesses had cost up to 152 billion do llar a year
includ ing the costs of med ica l bills lost wages and lost produc tivi ty (Smii middot 2010) Thus
food safety is cun-entl y an increasing important pub lic health issue laquougovenunents all over
the world are intensify ing the ir efforts to improve food safety
Among the majo r foodbome illness caused by mi croorgan ism are Salmonellosis
Campylobacteriosis Listeriosis and infectio ns due to entero haemonha gic (caJing intes tinal
bleeding) E coli (WHO 2007) III recent years Vibriosis had emerged to be an impOItant
bac te rial disease that canses human intestinal and ext ra-intestinal infections a ll over the world
(Chan elol 2002 Garc ia el al 2009 and Harth et al 2009) The three Vibrio species o f
primary significance in foodbo rne illnesses are V cholerae (serogroups 01 non-Ol and
2
- bull i
0 139) V vulnijiclIs and V paralwemolyricus Presently gast roenteitis caused by V
parahaemolyticus is extensively documented due to its increasing outbreaks (Alam el 01
2003 Gwendelynne el 01 2005 Gorny 2006 Harth et 01 2009 Roque et 01 2009)
V parahaemolyriclls is widely present in brackish and marine waters and infection to
human is regula rl y associated with the consumption of contaminated raw or undercooked
scafood (Guoxia ng el 01 2009) Ea rli er studies also demonstrated that V parahoemolylicus
can be easily iso lated from costal water sediments suspended particles and plankton due to
its halophi lic characteristic (Marlina el 01 2007 Zinuneman el 01 2007 Zu lkifli el al
2009 Vimala et 01 2010) However recent foodbome outbreaks throughout the world have
been intensive ly linked to consu mption of fresh fruits vegetables and unpasteurized juices
(Gorny 2006) Most recently Tunu ng et 01 (20 10) had rep0l1ed th at the vegetable samples
from supermarkets and wet markets in Selangor Malaysia were posit ive for pathogeni c
species of V parahaemolylicls Conversely there is no ot her known documented research on
the prevalence of this species in vegetables
In this st udy vegetables from two wet markets and two supem1arkets in Kuching
Sa rawak were sampled for the presence of V parallOemolyliclis The sa moles underwent
selective enrichment with alkaline peptone water (APW) supp lemented with 3 NaCI Then
presumptive V parahaemolYlicus were iso lated using TeES agar and CH ROMagar VibrioT
Following isolat ion gram stai ning together with a series of biochemica l tests were perfonned
to assist in the identification and confirmation of the iso lates
3
- bull i ~
IZ Objectives
This study was underta ken with the fo llowing objecti ves
I To isolate presumptive V parahaemolYlicus from marketed vegetabl es samples
in Kuching Sarawak by using Te BS and CHROMagar VibrioTM
II To confirm th e suspected V parahaemolyficus iso lates from marketed
vegetables samples in Kuching Sarawak by usi ng Gram stain and biochemical
tests
4
- bull i ~
USI KhJtlmal Makumat U umk VMVl~m ItLAYSlA AAAWAK
CHAPTER 2
LITERATURE REVIEW
21 The genus Vibrio
The genus Vibrio belongs to the fami ly Vibrionaceae and it consists of at least 34 recogn ized
species (HPA 2007a) In general Vibrio spp are Gram-negat ive fac ulta tively anaerobic
mOlile curved rods bacteria with a single polar flagellum (FEHD 2005) In surface waters of
the world Vibrios are among the mos t common surface organisms (Adeleye et aI 20 J 0)
They are present in both marine and fresh wa ter habiwts and commonly associa ted with
aquatic animals Vibrio pp such as V choerae V parahaemoylicus V vu nificus V
aginoylieus V mimicus V luvia lis V filmissii V melselmikovii V tollisae and V
dalltsea are common human pathogens (Tison 1999 Fi nkelstein 2000) T hey account for a
significant proportion of human infections as most of these vibrios secrete enterotoxins to
foods water or in the gastrointestinal tract (Nishibuchi and DePao la 2005)
22 Vibrio paralJlell1oytictls
Vibrio parahaemoy licus which is formerly known as Pasteurella parahaeliloytlca is a
non-sporing Gram-negat ive facu ltatively-anaerobic bacterium (Curti s el 01 2003) This
5
- ~
halophilic bacterium inhabits warm estuarine waters world wide (Bilung el al 2005) Thus il
reqlllres salt to survive and appears in high er concentrations during the warmer summer
months
The optimum temperature fo r the grow th of V parahaemolylicus is 37 degC and at Ihis
optimum temperature the bac terium has generation times of8 to 9 minutes in water and 12 [ 0
18 minutes in seafood (Montville and Matthews 2008) Thi s bacterium is the most IKeiy
Vibrio species to be imp licated in food borne diseases although both V vulnificus and V
cholera may also causes food bom e infections (Lawley e l al 2008) Clinical isolates of V
parrhaemolyticus produce beta type hemolysis on blood aga r (Wagatsuma agar) The
hemolysis is ca ll ed the Kanagawa-phenomenon (KP) which is associated with the production
ofa thermo stabl e d irect hemolys inTOH (Boyd 2008) TOH caused damage to the euka ryotic
cells by acting as a pore fonning toxin that alters the ion balance of cells (Boyd 2008)
Figure 21 SEM observa tlOos of V plrh cm)JJ(cu~
Image source Belas amp Colwe ll ( 1982)
6
-- UI
23 Fresh vegetables
Consumpti on of raw vegetables is common throughout the world not onl y owing to Ihe health
benefit but they are thought to be free from disease causing pathogens However recent food
borne outbreaks worldwi de is associ ated to consumption of fresh frui ts uncooked vegetables
and unpasteurized juices (Gorny 2006) Fruits and vegetable especially those that are eaten
raw without peeling can be the vehicles for transmission of a wide range of mi croorganis m
(Erdogru l and Sener 2005)
Based on Haeton and Jones (2008) E coli Salmonella spp and Listeria spp are
commonly present in vegetable samples Meldrum el 01 (2009) report ed that the two recent
large ou tbreaks in the United Kingdom showed signi fi cant health problems that cou ld ari se
from consumption of contaminated salad Another study of the preva lence of pathogens in
vegetables was reported by Little el 01 (2007) in which they found Listeria monocylOgenes in
ready-to-eat mixed salads in the United Kingdom Several other studi es on d ifferent
microorga nism isolated from vegetabl es were also report ed recent ly by local researchers
(Chai el 01 2008 Han el 01 2009 Ponni ah el 0 2009)
In Mala ys ia the presence of V parahoemo yticus has also been repOIt ed in raw
vegetables samples from wet markets and supeLl11arkets in Selangor (Tunung et 0 20 I 0)
This same study discovered that raw vegetabl es such as OenGnthe st l7i(era (Japanese
parsley) and C[cumis sali vus (Cucumber) were contaminated with virulent V
7
parahaemoiYlicus Consequen tly these vegetables could act as a transmission route and
poses risk to consumers after consumption Tunung el 01 (2011) had also demonstrated that
the vegetable samples fro m wet market contained higher levels of V paraiJaemolylicls as
compared w ith supennarkets The research suggested that there are many factors that can
contri bu te to the distribution of V parahaemoiylicus such as temperature holding ti me as
well as hygiene and sanitation practice (Tunung el aI 2011) However there is no known
documented research on V parahaemolYlicus in raw vegetables in Sarawak
24 Clinical characteristic of V parahaelllolyliclis infections
II parahaemoiyticus causes three distinct illness syndromes which are gastroenteritis wound
or soft tissues infections and septicemia (Sillljee 2007) The most freq uen t syndrome is
gastroeuteritis The symptoms include dianhea with abdomina l cramps nausea vo miting
headache and low-grade fever (S tarlin 2005 WilU1 and Konema n 2006 Montville and
Matthew 2008) Sometimes the diarrhea is bloody w ith stools described as meat washed
since tl1e stool is reddish and watery (Qadri el aI 2009)
As for wound or tis sue in fections this coudition occurs by the exposure of broken skin
or wound to warm seawater and seafood dripp ings (Ralph and Currie 2007 Simjee 2007)
In severe cases serious wound infection will resu lt in necrotizing fasc iiti s (Ralph and Curri e
2007) Although less common in V paraiwemolyliclIs infection septicemia is characteri7cd
by fever or hypotension with the isolation of V parahaemolyticus in the blood Septicemia
typicall y occurs in person with underlying health problems such as liver disease diabetes
cancer or other immune disorder (Simjee 2007) V parahaemoyticlis can even ca use fala l
septicemia in an immune-compromised hosts
The symptoms usuall y occur on the average of 15 hours but can began as early as 4
hours and as late as 36 hours after exposure and this will continue up to 3 days (Barceloux
200S) However these illnesses are usua lly categorized as a short illnesses and no prolonged
enect will take place after recovery (Wright et al 2009)
25 Isolation of V parahael1lolyliclIs
For the isolation of V parahaemoyticus an initial emichment using alka line peptone wat er
(APW) with pH S5 would be useful as this medium supports the growth uf viable ce lls
(Simjee 2007) Plati ng of emichment broth onto TCBS agar has been widely used for
selective isolation of V parahaemolytics from foods (Thompson el ai 2006 Oi Pinto et al
201 0 Canizalez -Roman et ai 2011 ) An average of 24 hours incubation at 37 degC is requ ired
fo r typica l V parahaemolylicus to develop colonies that are smooth gree n (s u~rose nega tive)
and 2 to 3 nun in di ameter on TCBS (ISO 2007) However V parahaenlOlytlcus co lonies on
TCBS agar are difficult to distinguish vi suall y from other bacterial colonies since they can be
covered by yellow color produced by sucrose-fennenting bacteria (Thompson el 01 2006
Rambach 2007)
9
_ ------~-- i ~~
Another widely used selective medium is CHROMagar VibrioT On this medium V
pllrahaemoiy liclis will developed mau ve color colonies (Kudo et aI 2001 Oi Pi nto 2010
Canizalez-Roman 2011) This agar medium contains substrates for beta-galactosidase which
was developed specifically to differentiate V parahaemolytics from ot her bacteria by us ing a
chromogenic su bstrate instead of sugar fermentation in traditional growth med ia such as
TCBS (Hara-Kudo et 01 2001) Therefore it is more sensitiv e and accurate for identi fying V
porahllemolyticlIs in food samples rather than the use of Te ES (Oi Pinto 2010 Canizalez-
Roman 20 II)
26 Identification through Gram-stain and biochemical tests
V parahaemoiyticus is a Gram-negative bacterium th us this organi sm stained pink or red
after Gram staining (HPA 20073) Under microscopic observation a typical V
pllrahaemolyticus appeared as rod characterist ica lly curved or comma-shoped The bacterium
is also positi ve for motility under microscopic observations (ISO 2007 BPA 2007a)
V parahaemolylieus ca n be identified through a seri es of biochemical tests According
to Khan et 01 (2007) V para haemolyticus can be distinct ively differentia ted fro m the other
Vibrio spp by performing sa lt tolerance and acid fro m femlen tation tests A typical V
parahaemoiYlicus co lonies would not grow in AFW without th e presence of at least 3 of sali
10
~-------
but it can only endure the presence of salt up to 8 (ISO 2007 Khan el aI 2007) In the
other hand the fermentation test for acid production is the most important test to differentiate
between V parahaemolyliclIs and V vulnificus species (Khan el aI 2007) Jayasinge et ai
(2010) even suggested that the combination of salt toleranc e test Voges-Proskauer test and
ONPG test can provide a strai ghtforward differentiation of V parahaemoly liclis from the
other Vibrionaceae species (Figure 31)
Citrate test screens the ab ilit y of bacterial isolates to utilize ci trate as its carbon and
energy source (MacFaddin 2000) This test is a very important test to identify gram-negative
pathogens (Tang el aI 1998) The MR-VP medium which was used for Met hyl red and
Voges Proskauer test contains glucose and peptone Methyl red test screens for the bacterial
metaboli sm pathways for glucose fermentation while the Voges-Proskauer tes t is an assay for
organisms which ferment glucose with only one fermentation product usuall y acetic acid
The TS[ test is another useful test tha t can distinguish V parahaemolyticus from other
bacteria (ISO 2007) The TSJ agar med ium contains peptone fenous sulfate lactose sucrose
and a small amount of glucose (dex trose) The pH indi cator is phenol red This medium
differentiates bacteria based on their ability to fennent glucose lactose ando r sucrose and on
their abi lity to reduce sulfur to hydrogen sulfide (H S) The T SJ test also haj the abi lity to
determine the production of H]S (Kim et aI 1999)
In additi on other bi ochemica l tests such as oxidase lysine decarboxylase om ithi ne
decarboxylase ca talase and indole can also be used for the purpose of identifying V
II
-- ---- - ----------- - ~i
parahaemoyticus iso lates (ISO 2007 Jayasinghe et ai 2007 Khan et aI 2007) The
biochemical reactions o f V parahaemoiyticus are summarized in Table 21
Table 21 Siandard biochemical tesl reactions of V porahaemoyticus
TeSi S Reaction of V parahaem olyaclIs Refe rences Salt tol ance
+
Sali ne TSI agar KIA
No HS and gas production
Simmons Citrnh
Voges-Proskauer
Methyl-Red +
[SO (2007)
Jayasinge et al (2007)
Khan et of (2007)
ISO (2007)
Kim et I (1999)
Abide et til (1999)
Kim 11111 (1999)
Khan et til (2007)
Jayasinge el III (20 (0)
Kim et 0 (2 007)
Note (-) ~ nega live (+) ~ positi ve K ~ alka line ~ aC idic
12
CHAPT ER 3
MATERIALS AND METI IODS
31 Samples collection
Eighty raw vegetable samples were collected week ly from two wet markets (WI amp W2) and
two supennarkets (S 1 amp S2) in Kuching Sarawa k The collected samples consisted o f four
di fferent groups of vegetables which are regularly consumed ra w by the locals These samples
wcr~ rand omly selected within ten weeks -period which is from October 20 10 to January
20 11 Duri ng collection each sample was transferred to clean plastic bags labeled acco rding
to the place they were collected and were analyzed immed iately on arriva l to the labo rat ory
The types of raw vegetable sa mples obtained were as shown in Table 31
Tab le 31 Types o f raw vegetable samples co llected from wet markes and supcmlarkels
Group Loca l nam e English nam e Scienti fi c Home l mple coding No or samples
Soil Kubis Cabbage Brassica v eracea K 20 Roots Lobak Canmiddotot Douclfs carola C ~O
mera h Beans KaCtlO g Four-winged PsophOCaJjJ lls KB cO
bow l bean JeIlagulIolobus Shoot Tauge Bean-I_prout Vigna rad mc T 10 Total 80
13
---------~ I I
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
bullbull
TABLE OF CONTENTS
ACKN OWLEI)GEMENT
DECLARATJO II
TABLE OF CONTENTS I II
LIST OF ABBREVIATIONS v
LIST OF TABLES D FIGURES V I
ABSTRACT I ABSTRAK
CHAPTER 1
CHAPTER 2
CH PTER3
INTRODUCTION 2
Lrt ERATlJRE REVIEWS 5
21 The genus Vibrio 5
22 Vibrio parahaemolylicus 5
23 Fresh vegetables 7
24 Clinica l cha ract eristics of V parahaemolyticus 8
infections
25 Iso lation of V parahaemolyticus 9
26 Id entilication through Gra m-Stain and biochemical 10
tes ts
JllATERlAL AND METHODS 13
31 Samples co ll ection 13
32 Samples preparat ion and enrichment 14
III
__a ------ _bull 1 shy
33 Isolation o f V parahaemolylicus 14
34 Preparation for Gram-stain and biochemical tests 15
35 G ram slain prolocol 16
36 Biochemicallesls 16
36 1 Voges-Proskauer test 18
362 Methyl-Red test 18
363 Sa lt tolerance test IS
36 4 Triple sugar iron test (TSI) 19
365 Hyd rogen sulfid e and gas production 19
366 Ci trate test 19
RESULTS 20
41 Iso lation of V parahaemolyticlIs 20
43 Gram-stai n and biochemical tests 23
CHAPTER 5 DI CUSSION 28
51 Vegetab les types 28
52 Co loni es appearance on selective aga r 28
53 Isolation rate of V paraiaemolylicus on TCa and 30
C HROMagar Vibrio
54 In terpre ta tions of biochemical tests 34
55 Preva lence of V parahaemolylicus 35
CIIAPTER 6 CONCLUS ION 37
CHAPTER 7 REFERENCES 39
IV
-shy
--
C
APW
g
KJA
NK
1111
mm
MR
NaCI
PCR
Spp
TeBS
TSI
VP
V parahaemoLytiCLIs Vp
WHO
LIST OF ABBREVIATIONS
percentage
degree celcius
Alkaline Peptone Water
gram
hyd rogen sulfite
alkaline slant acidic bUlt
acidic slant alkaline butt
microliter
milli liter
milimeter
Methyl-red
Sodium chloride
Polymerase Chain Reaction
species
Tiosulphate Citrate Bile Salt Sucrose
Tliple Sugar Iron
V oges-Proskaeur
Vibrio parallOemolyticlIs
World Heal th Organizati ons
v
~-
LIST OF TABLES
Tables Page
Table 21 Standard biochemical test reactio ns of V parahaemolyliclIs 12
Table 31 Types of raw vegetable samples collected from wet markets and 13
supermarkets
Table 41 Results for the presence of V parahaemolyticus on TCBS and 2 1
CHROMa gar Vibrio from the vegetable sa mples
Table 42 Result s of microscopic observations of the suspected V 27
parahaemolylicus iso lates
Table 43 Overall resuit of biochemical tests 24
vi
--bull i~
LIST OF FIG URES
Figures Page
Figurc 2t SEM observation o f Ii parahaemolyticus Image source Belas amp 6
Colwell (1 982)
and C I-IR OM agar Vib rio trade based on different sam pling locr io ns
Figure 31 Biochemical keys fo r the identification o f Vibrio species 17
Figure 41 The isolation o f Ii parQhaemolyticus on TCBS 22
Figure 42 Appearance of mixed colonies on CHROM agar Vib riotrade 22
Figure 43 Pure mauve co lonies obtained a fter sub -culturing 23
figurc 44 Reac tio ns of (a) B7KBS and (b) B8CW towards Voges -Proskauer test 24
Figure 45 Reactio ns of (a) B7KBS and (b) BI CW towards M dhly-red test 25
Figure 46 The reaction of a ll isolates towards citrate test 25
Figure 47 The reaction o f (a) B7KBS = KJA and (b) B3KW = AK fo r TSltes t 26
Figure 48 The reactio n o fB7KBS towards (a) 0 NaC I and (b) 8 Nae 27
Figu re 5 1 Bar graph o f the di stribution of presumptive pas iti ve sampl es on TCBS 30
Figure 52 Pie chart o f the distribu tion of presumptive Ii paraliaemolyliclls 31
between different vegetable types on CHROM aga r Vibrio
Figure 53 Pi e chart of the distribution of presumptive Ii parahaemolylicus 33
between di fferent vegetab le types on CHROMagar Vibriotrade
VII
--~bull i lj
Tbe Incidence of Vibrio p arahaellloly ticlis in Raw Vegetables at Local Wet Markets and Supermarkets in Kuching Sarawak
Sabatini lihob
Resource BIOIClhnoltlg) Progrtlm me Facu Ity or Resource Science Bud T~lln(llogy
Uni versity Malaysb Sa rawa k
ABSTRACT
Vibrio parawemoyliCLls is a foodbome pa thogen that is regular ly assoc iated with the consu mption o f
raw or undercooked seafood The mos t frequent sympto m of V parahaemoylicus infecr ion is
gastroenteritis Recen tly this pa thogen was also disco vered in raw vegetable samples in Peninsular
Malaysia Thus the aim of this study is to isolate and de tect V paahaemoylicu~ in raw vegdables T he samples which consisted of carrot fourmiddot winged bean cabbage and bea n sp ro ul were purc hased
tiom two wet markets and two supenn arkets in Kucll ing Sarawak In th is stud y alkaline peplone
water (A PW) wa s used as the enridunent medium The combinati o n o f two types o f selective agar
TCBS and CHROMa gar Vibrio nbull1 was successfully app lied to isolate the bacteria A fte r a series of
biochemical tests only 2 out of the 80 samples were pos itive for the p resence of V parahaemoylicLls
Although the peroentage of V pOrehaemolylics prevalence was low (2 5) the resu lts h ighli ghted
the fac t that raw vege tables could also be contaminated w It h V parahaenro~Vlicus
Key words Vib rio parahemotyfic~ raw vegetables TC BS and CHROMagar Vibrio rM
ABSTRAK
Vibrio parawcmoylicus merupakan sejenis patogen bmvaan makanafl )long sering dikairkan dengan
pengambiall makonon laul seewa men fa h Tonda paling kemp bagi j angkitall k parahwnJ r lvticlfs
ialah gasroenlritis TerbolU pafogen ini Ina dijumpa i di dalam sUlllp~1 scJyuran melJfah di
Semenanjung A1nlaYiia Kajial1 ini berlujuan un uk mengasmg dan mengesan V nanlhaemolliqi di
daall1 sampel $oyuran menfah Sampel yang lerdiri doripada obok merah kac(mg botol kobis dan
(l uge teah dlbeli dari dU(J pasai basah Clnd clua pasar raya di Kuchi lg Sorm lak Il l pepton beralktrli
(A PW) fel(lh digul1akan Ill1tuk memperbonyakko1 jwn Jah bakfcna Kombilw si pelggunaon agar
terpilih Tess dan CHROkiagar Vibrio T1 tdoh bejaya mel11ellcilkon ba( ur ia yallg dikehendoki
Selelah beberapa siri lljion biokimia didapafi 2 daripada 80 sampel relah dlcemQj oleh 1 rar(fhoenlJliJji(u~ Mcsklpun perafus kehadiran V norahaemolvticus adalah remJah (25~ o)
kepuflfsan kajian ini menunjukkan bahawo saYllran mental boleh dicemari ole Y paraha~moh IIcm
Kata klllt ci Vibrio oorqhaemol liclfs sayuran men fah TCBS dOll CHROtviagar Vihriotrade
-
CHAPTER 1
INTRODUCTION
11 Introduction
Foodbome illness assoc iated with consumption of contam inated food is a corrunon hea lth
problem that occurs worldwide The global inc idence of food bo rne ill ness is d ifficu lt to
estimate but it has been reported that in 2005 alone around 76 million cases of food bo rne
diseases resu lting in 325000 hospita liza tions and 5000 dea ths are es timated to occur in the
United State (WHO 20 11 ) In addition food contaminat ion also causes enormous socia l and
economical burden on the community A consumer research repon released on 2010 revea led
that the healt h-related costs of food-borne illnesses had cost up to 152 billion do llar a year
includ ing the costs of med ica l bills lost wages and lost produc tivi ty (Smii middot 2010) Thus
food safety is cun-entl y an increasing important pub lic health issue laquougovenunents all over
the world are intensify ing the ir efforts to improve food safety
Among the majo r foodbome illness caused by mi croorgan ism are Salmonellosis
Campylobacteriosis Listeriosis and infectio ns due to entero haemonha gic (caJing intes tinal
bleeding) E coli (WHO 2007) III recent years Vibriosis had emerged to be an impOItant
bac te rial disease that canses human intestinal and ext ra-intestinal infections a ll over the world
(Chan elol 2002 Garc ia el al 2009 and Harth et al 2009) The three Vibrio species o f
primary significance in foodbo rne illnesses are V cholerae (serogroups 01 non-Ol and
2
- bull i
0 139) V vulnijiclIs and V paralwemolyricus Presently gast roenteitis caused by V
parahaemolyticus is extensively documented due to its increasing outbreaks (Alam el 01
2003 Gwendelynne el 01 2005 Gorny 2006 Harth et 01 2009 Roque et 01 2009)
V parahaemolyriclls is widely present in brackish and marine waters and infection to
human is regula rl y associated with the consumption of contaminated raw or undercooked
scafood (Guoxia ng el 01 2009) Ea rli er studies also demonstrated that V parahoemolylicus
can be easily iso lated from costal water sediments suspended particles and plankton due to
its halophi lic characteristic (Marlina el 01 2007 Zinuneman el 01 2007 Zu lkifli el al
2009 Vimala et 01 2010) However recent foodbome outbreaks throughout the world have
been intensive ly linked to consu mption of fresh fruits vegetables and unpasteurized juices
(Gorny 2006) Most recently Tunu ng et 01 (20 10) had rep0l1ed th at the vegetable samples
from supermarkets and wet markets in Selangor Malaysia were posit ive for pathogeni c
species of V parahaemolylicls Conversely there is no ot her known documented research on
the prevalence of this species in vegetables
In this st udy vegetables from two wet markets and two supem1arkets in Kuching
Sa rawak were sampled for the presence of V parallOemolyliclis The sa moles underwent
selective enrichment with alkaline peptone water (APW) supp lemented with 3 NaCI Then
presumptive V parahaemolYlicus were iso lated using TeES agar and CH ROMagar VibrioT
Following isolat ion gram stai ning together with a series of biochemica l tests were perfonned
to assist in the identification and confirmation of the iso lates
3
- bull i ~
IZ Objectives
This study was underta ken with the fo llowing objecti ves
I To isolate presumptive V parahaemolYlicus from marketed vegetabl es samples
in Kuching Sarawak by using Te BS and CHROMagar VibrioTM
II To confirm th e suspected V parahaemolyficus iso lates from marketed
vegetables samples in Kuching Sarawak by usi ng Gram stain and biochemical
tests
4
- bull i ~
USI KhJtlmal Makumat U umk VMVl~m ItLAYSlA AAAWAK
CHAPTER 2
LITERATURE REVIEW
21 The genus Vibrio
The genus Vibrio belongs to the fami ly Vibrionaceae and it consists of at least 34 recogn ized
species (HPA 2007a) In general Vibrio spp are Gram-negat ive fac ulta tively anaerobic
mOlile curved rods bacteria with a single polar flagellum (FEHD 2005) In surface waters of
the world Vibrios are among the mos t common surface organisms (Adeleye et aI 20 J 0)
They are present in both marine and fresh wa ter habiwts and commonly associa ted with
aquatic animals Vibrio pp such as V choerae V parahaemoylicus V vu nificus V
aginoylieus V mimicus V luvia lis V filmissii V melselmikovii V tollisae and V
dalltsea are common human pathogens (Tison 1999 Fi nkelstein 2000) T hey account for a
significant proportion of human infections as most of these vibrios secrete enterotoxins to
foods water or in the gastrointestinal tract (Nishibuchi and DePao la 2005)
22 Vibrio paralJlell1oytictls
Vibrio parahaemoy licus which is formerly known as Pasteurella parahaeliloytlca is a
non-sporing Gram-negat ive facu ltatively-anaerobic bacterium (Curti s el 01 2003) This
5
- ~
halophilic bacterium inhabits warm estuarine waters world wide (Bilung el al 2005) Thus il
reqlllres salt to survive and appears in high er concentrations during the warmer summer
months
The optimum temperature fo r the grow th of V parahaemolylicus is 37 degC and at Ihis
optimum temperature the bac terium has generation times of8 to 9 minutes in water and 12 [ 0
18 minutes in seafood (Montville and Matthews 2008) Thi s bacterium is the most IKeiy
Vibrio species to be imp licated in food borne diseases although both V vulnificus and V
cholera may also causes food bom e infections (Lawley e l al 2008) Clinical isolates of V
parrhaemolyticus produce beta type hemolysis on blood aga r (Wagatsuma agar) The
hemolysis is ca ll ed the Kanagawa-phenomenon (KP) which is associated with the production
ofa thermo stabl e d irect hemolys inTOH (Boyd 2008) TOH caused damage to the euka ryotic
cells by acting as a pore fonning toxin that alters the ion balance of cells (Boyd 2008)
Figure 21 SEM observa tlOos of V plrh cm)JJ(cu~
Image source Belas amp Colwe ll ( 1982)
6
-- UI
23 Fresh vegetables
Consumpti on of raw vegetables is common throughout the world not onl y owing to Ihe health
benefit but they are thought to be free from disease causing pathogens However recent food
borne outbreaks worldwi de is associ ated to consumption of fresh frui ts uncooked vegetables
and unpasteurized juices (Gorny 2006) Fruits and vegetable especially those that are eaten
raw without peeling can be the vehicles for transmission of a wide range of mi croorganis m
(Erdogru l and Sener 2005)
Based on Haeton and Jones (2008) E coli Salmonella spp and Listeria spp are
commonly present in vegetable samples Meldrum el 01 (2009) report ed that the two recent
large ou tbreaks in the United Kingdom showed signi fi cant health problems that cou ld ari se
from consumption of contaminated salad Another study of the preva lence of pathogens in
vegetables was reported by Little el 01 (2007) in which they found Listeria monocylOgenes in
ready-to-eat mixed salads in the United Kingdom Several other studi es on d ifferent
microorga nism isolated from vegetabl es were also report ed recent ly by local researchers
(Chai el 01 2008 Han el 01 2009 Ponni ah el 0 2009)
In Mala ys ia the presence of V parahoemo yticus has also been repOIt ed in raw
vegetables samples from wet markets and supeLl11arkets in Selangor (Tunung et 0 20 I 0)
This same study discovered that raw vegetabl es such as OenGnthe st l7i(era (Japanese
parsley) and C[cumis sali vus (Cucumber) were contaminated with virulent V
7
parahaemoiYlicus Consequen tly these vegetables could act as a transmission route and
poses risk to consumers after consumption Tunung el 01 (2011) had also demonstrated that
the vegetable samples fro m wet market contained higher levels of V paraiJaemolylicls as
compared w ith supennarkets The research suggested that there are many factors that can
contri bu te to the distribution of V parahaemoiylicus such as temperature holding ti me as
well as hygiene and sanitation practice (Tunung el aI 2011) However there is no known
documented research on V parahaemolYlicus in raw vegetables in Sarawak
24 Clinical characteristic of V parahaelllolyliclis infections
II parahaemoiyticus causes three distinct illness syndromes which are gastroenteritis wound
or soft tissues infections and septicemia (Sillljee 2007) The most freq uen t syndrome is
gastroeuteritis The symptoms include dianhea with abdomina l cramps nausea vo miting
headache and low-grade fever (S tarlin 2005 WilU1 and Konema n 2006 Montville and
Matthew 2008) Sometimes the diarrhea is bloody w ith stools described as meat washed
since tl1e stool is reddish and watery (Qadri el aI 2009)
As for wound or tis sue in fections this coudition occurs by the exposure of broken skin
or wound to warm seawater and seafood dripp ings (Ralph and Currie 2007 Simjee 2007)
In severe cases serious wound infection will resu lt in necrotizing fasc iiti s (Ralph and Curri e
2007) Although less common in V paraiwemolyliclIs infection septicemia is characteri7cd
by fever or hypotension with the isolation of V parahaemolyticus in the blood Septicemia
typicall y occurs in person with underlying health problems such as liver disease diabetes
cancer or other immune disorder (Simjee 2007) V parahaemoyticlis can even ca use fala l
septicemia in an immune-compromised hosts
The symptoms usuall y occur on the average of 15 hours but can began as early as 4
hours and as late as 36 hours after exposure and this will continue up to 3 days (Barceloux
200S) However these illnesses are usua lly categorized as a short illnesses and no prolonged
enect will take place after recovery (Wright et al 2009)
25 Isolation of V parahael1lolyliclIs
For the isolation of V parahaemoyticus an initial emichment using alka line peptone wat er
(APW) with pH S5 would be useful as this medium supports the growth uf viable ce lls
(Simjee 2007) Plati ng of emichment broth onto TCBS agar has been widely used for
selective isolation of V parahaemolytics from foods (Thompson el ai 2006 Oi Pinto et al
201 0 Canizalez -Roman et ai 2011 ) An average of 24 hours incubation at 37 degC is requ ired
fo r typica l V parahaemolylicus to develop colonies that are smooth gree n (s u~rose nega tive)
and 2 to 3 nun in di ameter on TCBS (ISO 2007) However V parahaenlOlytlcus co lonies on
TCBS agar are difficult to distinguish vi suall y from other bacterial colonies since they can be
covered by yellow color produced by sucrose-fennenting bacteria (Thompson el 01 2006
Rambach 2007)
9
_ ------~-- i ~~
Another widely used selective medium is CHROMagar VibrioT On this medium V
pllrahaemoiy liclis will developed mau ve color colonies (Kudo et aI 2001 Oi Pi nto 2010
Canizalez-Roman 2011) This agar medium contains substrates for beta-galactosidase which
was developed specifically to differentiate V parahaemolytics from ot her bacteria by us ing a
chromogenic su bstrate instead of sugar fermentation in traditional growth med ia such as
TCBS (Hara-Kudo et 01 2001) Therefore it is more sensitiv e and accurate for identi fying V
porahllemolyticlIs in food samples rather than the use of Te ES (Oi Pinto 2010 Canizalez-
Roman 20 II)
26 Identification through Gram-stain and biochemical tests
V parahaemoiyticus is a Gram-negative bacterium th us this organi sm stained pink or red
after Gram staining (HPA 20073) Under microscopic observation a typical V
pllrahaemolyticus appeared as rod characterist ica lly curved or comma-shoped The bacterium
is also positi ve for motility under microscopic observations (ISO 2007 BPA 2007a)
V parahaemolylieus ca n be identified through a seri es of biochemical tests According
to Khan et 01 (2007) V para haemolyticus can be distinct ively differentia ted fro m the other
Vibrio spp by performing sa lt tolerance and acid fro m femlen tation tests A typical V
parahaemoiYlicus co lonies would not grow in AFW without th e presence of at least 3 of sali
10
~-------
but it can only endure the presence of salt up to 8 (ISO 2007 Khan el aI 2007) In the
other hand the fermentation test for acid production is the most important test to differentiate
between V parahaemolyliclIs and V vulnificus species (Khan el aI 2007) Jayasinge et ai
(2010) even suggested that the combination of salt toleranc e test Voges-Proskauer test and
ONPG test can provide a strai ghtforward differentiation of V parahaemoly liclis from the
other Vibrionaceae species (Figure 31)
Citrate test screens the ab ilit y of bacterial isolates to utilize ci trate as its carbon and
energy source (MacFaddin 2000) This test is a very important test to identify gram-negative
pathogens (Tang el aI 1998) The MR-VP medium which was used for Met hyl red and
Voges Proskauer test contains glucose and peptone Methyl red test screens for the bacterial
metaboli sm pathways for glucose fermentation while the Voges-Proskauer tes t is an assay for
organisms which ferment glucose with only one fermentation product usuall y acetic acid
The TS[ test is another useful test tha t can distinguish V parahaemolyticus from other
bacteria (ISO 2007) The TSJ agar med ium contains peptone fenous sulfate lactose sucrose
and a small amount of glucose (dex trose) The pH indi cator is phenol red This medium
differentiates bacteria based on their ability to fennent glucose lactose ando r sucrose and on
their abi lity to reduce sulfur to hydrogen sulfide (H S) The T SJ test also haj the abi lity to
determine the production of H]S (Kim et aI 1999)
In additi on other bi ochemica l tests such as oxidase lysine decarboxylase om ithi ne
decarboxylase ca talase and indole can also be used for the purpose of identifying V
II
-- ---- - ----------- - ~i
parahaemoyticus iso lates (ISO 2007 Jayasinghe et ai 2007 Khan et aI 2007) The
biochemical reactions o f V parahaemoiyticus are summarized in Table 21
Table 21 Siandard biochemical tesl reactions of V porahaemoyticus
TeSi S Reaction of V parahaem olyaclIs Refe rences Salt tol ance
+
Sali ne TSI agar KIA
No HS and gas production
Simmons Citrnh
Voges-Proskauer
Methyl-Red +
[SO (2007)
Jayasinge et al (2007)
Khan et of (2007)
ISO (2007)
Kim et I (1999)
Abide et til (1999)
Kim 11111 (1999)
Khan et til (2007)
Jayasinge el III (20 (0)
Kim et 0 (2 007)
Note (-) ~ nega live (+) ~ positi ve K ~ alka line ~ aC idic
12
CHAPT ER 3
MATERIALS AND METI IODS
31 Samples collection
Eighty raw vegetable samples were collected week ly from two wet markets (WI amp W2) and
two supennarkets (S 1 amp S2) in Kuching Sarawa k The collected samples consisted o f four
di fferent groups of vegetables which are regularly consumed ra w by the locals These samples
wcr~ rand omly selected within ten weeks -period which is from October 20 10 to January
20 11 Duri ng collection each sample was transferred to clean plastic bags labeled acco rding
to the place they were collected and were analyzed immed iately on arriva l to the labo rat ory
The types of raw vegetable sa mples obtained were as shown in Table 31
Tab le 31 Types o f raw vegetable samples co llected from wet markes and supcmlarkels
Group Loca l nam e English nam e Scienti fi c Home l mple coding No or samples
Soil Kubis Cabbage Brassica v eracea K 20 Roots Lobak Canmiddotot Douclfs carola C ~O
mera h Beans KaCtlO g Four-winged PsophOCaJjJ lls KB cO
bow l bean JeIlagulIolobus Shoot Tauge Bean-I_prout Vigna rad mc T 10 Total 80
13
---------~ I I
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
33 Isolation o f V parahaemolylicus 14
34 Preparation for Gram-stain and biochemical tests 15
35 G ram slain prolocol 16
36 Biochemicallesls 16
36 1 Voges-Proskauer test 18
362 Methyl-Red test 18
363 Sa lt tolerance test IS
36 4 Triple sugar iron test (TSI) 19
365 Hyd rogen sulfid e and gas production 19
366 Ci trate test 19
RESULTS 20
41 Iso lation of V parahaemolyticlIs 20
43 Gram-stai n and biochemical tests 23
CHAPTER 5 DI CUSSION 28
51 Vegetab les types 28
52 Co loni es appearance on selective aga r 28
53 Isolation rate of V paraiaemolylicus on TCa and 30
C HROMagar Vibrio
54 In terpre ta tions of biochemical tests 34
55 Preva lence of V parahaemolylicus 35
CIIAPTER 6 CONCLUS ION 37
CHAPTER 7 REFERENCES 39
IV
-shy
--
C
APW
g
KJA
NK
1111
mm
MR
NaCI
PCR
Spp
TeBS
TSI
VP
V parahaemoLytiCLIs Vp
WHO
LIST OF ABBREVIATIONS
percentage
degree celcius
Alkaline Peptone Water
gram
hyd rogen sulfite
alkaline slant acidic bUlt
acidic slant alkaline butt
microliter
milli liter
milimeter
Methyl-red
Sodium chloride
Polymerase Chain Reaction
species
Tiosulphate Citrate Bile Salt Sucrose
Tliple Sugar Iron
V oges-Proskaeur
Vibrio parallOemolyticlIs
World Heal th Organizati ons
v
~-
LIST OF TABLES
Tables Page
Table 21 Standard biochemical test reactio ns of V parahaemolyliclIs 12
Table 31 Types of raw vegetable samples collected from wet markets and 13
supermarkets
Table 41 Results for the presence of V parahaemolyticus on TCBS and 2 1
CHROMa gar Vibrio from the vegetable sa mples
Table 42 Result s of microscopic observations of the suspected V 27
parahaemolylicus iso lates
Table 43 Overall resuit of biochemical tests 24
vi
--bull i~
LIST OF FIG URES
Figures Page
Figurc 2t SEM observation o f Ii parahaemolyticus Image source Belas amp 6
Colwell (1 982)
and C I-IR OM agar Vib rio trade based on different sam pling locr io ns
Figure 31 Biochemical keys fo r the identification o f Vibrio species 17
Figure 41 The isolation o f Ii parQhaemolyticus on TCBS 22
Figure 42 Appearance of mixed colonies on CHROM agar Vib riotrade 22
Figure 43 Pure mauve co lonies obtained a fter sub -culturing 23
figurc 44 Reac tio ns of (a) B7KBS and (b) B8CW towards Voges -Proskauer test 24
Figure 45 Reactio ns of (a) B7KBS and (b) BI CW towards M dhly-red test 25
Figure 46 The reaction of a ll isolates towards citrate test 25
Figure 47 The reaction o f (a) B7KBS = KJA and (b) B3KW = AK fo r TSltes t 26
Figure 48 The reactio n o fB7KBS towards (a) 0 NaC I and (b) 8 Nae 27
Figu re 5 1 Bar graph o f the di stribution of presumptive pas iti ve sampl es on TCBS 30
Figure 52 Pie chart o f the distribu tion of presumptive Ii paraliaemolyliclls 31
between different vegetable types on CHROM aga r Vibrio
Figure 53 Pi e chart of the distribution of presumptive Ii parahaemolylicus 33
between di fferent vegetab le types on CHROMagar Vibriotrade
VII
--~bull i lj
Tbe Incidence of Vibrio p arahaellloly ticlis in Raw Vegetables at Local Wet Markets and Supermarkets in Kuching Sarawak
Sabatini lihob
Resource BIOIClhnoltlg) Progrtlm me Facu Ity or Resource Science Bud T~lln(llogy
Uni versity Malaysb Sa rawa k
ABSTRACT
Vibrio parawemoyliCLls is a foodbome pa thogen that is regular ly assoc iated with the consu mption o f
raw or undercooked seafood The mos t frequent sympto m of V parahaemoylicus infecr ion is
gastroenteritis Recen tly this pa thogen was also disco vered in raw vegetable samples in Peninsular
Malaysia Thus the aim of this study is to isolate and de tect V paahaemoylicu~ in raw vegdables T he samples which consisted of carrot fourmiddot winged bean cabbage and bea n sp ro ul were purc hased
tiom two wet markets and two supenn arkets in Kucll ing Sarawak In th is stud y alkaline peplone
water (A PW) wa s used as the enridunent medium The combinati o n o f two types o f selective agar
TCBS and CHROMa gar Vibrio nbull1 was successfully app lied to isolate the bacteria A fte r a series of
biochemical tests only 2 out of the 80 samples were pos itive for the p resence of V parahaemoylicLls
Although the peroentage of V pOrehaemolylics prevalence was low (2 5) the resu lts h ighli ghted
the fac t that raw vege tables could also be contaminated w It h V parahaenro~Vlicus
Key words Vib rio parahemotyfic~ raw vegetables TC BS and CHROMagar Vibrio rM
ABSTRAK
Vibrio parawcmoylicus merupakan sejenis patogen bmvaan makanafl )long sering dikairkan dengan
pengambiall makonon laul seewa men fa h Tonda paling kemp bagi j angkitall k parahwnJ r lvticlfs
ialah gasroenlritis TerbolU pafogen ini Ina dijumpa i di dalam sUlllp~1 scJyuran melJfah di
Semenanjung A1nlaYiia Kajial1 ini berlujuan un uk mengasmg dan mengesan V nanlhaemolliqi di
daall1 sampel $oyuran menfah Sampel yang lerdiri doripada obok merah kac(mg botol kobis dan
(l uge teah dlbeli dari dU(J pasai basah Clnd clua pasar raya di Kuchi lg Sorm lak Il l pepton beralktrli
(A PW) fel(lh digul1akan Ill1tuk memperbonyakko1 jwn Jah bakfcna Kombilw si pelggunaon agar
terpilih Tess dan CHROkiagar Vibrio T1 tdoh bejaya mel11ellcilkon ba( ur ia yallg dikehendoki
Selelah beberapa siri lljion biokimia didapafi 2 daripada 80 sampel relah dlcemQj oleh 1 rar(fhoenlJliJji(u~ Mcsklpun perafus kehadiran V norahaemolvticus adalah remJah (25~ o)
kepuflfsan kajian ini menunjukkan bahawo saYllran mental boleh dicemari ole Y paraha~moh IIcm
Kata klllt ci Vibrio oorqhaemol liclfs sayuran men fah TCBS dOll CHROtviagar Vihriotrade
-
CHAPTER 1
INTRODUCTION
11 Introduction
Foodbome illness assoc iated with consumption of contam inated food is a corrunon hea lth
problem that occurs worldwide The global inc idence of food bo rne ill ness is d ifficu lt to
estimate but it has been reported that in 2005 alone around 76 million cases of food bo rne
diseases resu lting in 325000 hospita liza tions and 5000 dea ths are es timated to occur in the
United State (WHO 20 11 ) In addition food contaminat ion also causes enormous socia l and
economical burden on the community A consumer research repon released on 2010 revea led
that the healt h-related costs of food-borne illnesses had cost up to 152 billion do llar a year
includ ing the costs of med ica l bills lost wages and lost produc tivi ty (Smii middot 2010) Thus
food safety is cun-entl y an increasing important pub lic health issue laquougovenunents all over
the world are intensify ing the ir efforts to improve food safety
Among the majo r foodbome illness caused by mi croorgan ism are Salmonellosis
Campylobacteriosis Listeriosis and infectio ns due to entero haemonha gic (caJing intes tinal
bleeding) E coli (WHO 2007) III recent years Vibriosis had emerged to be an impOItant
bac te rial disease that canses human intestinal and ext ra-intestinal infections a ll over the world
(Chan elol 2002 Garc ia el al 2009 and Harth et al 2009) The three Vibrio species o f
primary significance in foodbo rne illnesses are V cholerae (serogroups 01 non-Ol and
2
- bull i
0 139) V vulnijiclIs and V paralwemolyricus Presently gast roenteitis caused by V
parahaemolyticus is extensively documented due to its increasing outbreaks (Alam el 01
2003 Gwendelynne el 01 2005 Gorny 2006 Harth et 01 2009 Roque et 01 2009)
V parahaemolyriclls is widely present in brackish and marine waters and infection to
human is regula rl y associated with the consumption of contaminated raw or undercooked
scafood (Guoxia ng el 01 2009) Ea rli er studies also demonstrated that V parahoemolylicus
can be easily iso lated from costal water sediments suspended particles and plankton due to
its halophi lic characteristic (Marlina el 01 2007 Zinuneman el 01 2007 Zu lkifli el al
2009 Vimala et 01 2010) However recent foodbome outbreaks throughout the world have
been intensive ly linked to consu mption of fresh fruits vegetables and unpasteurized juices
(Gorny 2006) Most recently Tunu ng et 01 (20 10) had rep0l1ed th at the vegetable samples
from supermarkets and wet markets in Selangor Malaysia were posit ive for pathogeni c
species of V parahaemolylicls Conversely there is no ot her known documented research on
the prevalence of this species in vegetables
In this st udy vegetables from two wet markets and two supem1arkets in Kuching
Sa rawak were sampled for the presence of V parallOemolyliclis The sa moles underwent
selective enrichment with alkaline peptone water (APW) supp lemented with 3 NaCI Then
presumptive V parahaemolYlicus were iso lated using TeES agar and CH ROMagar VibrioT
Following isolat ion gram stai ning together with a series of biochemica l tests were perfonned
to assist in the identification and confirmation of the iso lates
3
- bull i ~
IZ Objectives
This study was underta ken with the fo llowing objecti ves
I To isolate presumptive V parahaemolYlicus from marketed vegetabl es samples
in Kuching Sarawak by using Te BS and CHROMagar VibrioTM
II To confirm th e suspected V parahaemolyficus iso lates from marketed
vegetables samples in Kuching Sarawak by usi ng Gram stain and biochemical
tests
4
- bull i ~
USI KhJtlmal Makumat U umk VMVl~m ItLAYSlA AAAWAK
CHAPTER 2
LITERATURE REVIEW
21 The genus Vibrio
The genus Vibrio belongs to the fami ly Vibrionaceae and it consists of at least 34 recogn ized
species (HPA 2007a) In general Vibrio spp are Gram-negat ive fac ulta tively anaerobic
mOlile curved rods bacteria with a single polar flagellum (FEHD 2005) In surface waters of
the world Vibrios are among the mos t common surface organisms (Adeleye et aI 20 J 0)
They are present in both marine and fresh wa ter habiwts and commonly associa ted with
aquatic animals Vibrio pp such as V choerae V parahaemoylicus V vu nificus V
aginoylieus V mimicus V luvia lis V filmissii V melselmikovii V tollisae and V
dalltsea are common human pathogens (Tison 1999 Fi nkelstein 2000) T hey account for a
significant proportion of human infections as most of these vibrios secrete enterotoxins to
foods water or in the gastrointestinal tract (Nishibuchi and DePao la 2005)
22 Vibrio paralJlell1oytictls
Vibrio parahaemoy licus which is formerly known as Pasteurella parahaeliloytlca is a
non-sporing Gram-negat ive facu ltatively-anaerobic bacterium (Curti s el 01 2003) This
5
- ~
halophilic bacterium inhabits warm estuarine waters world wide (Bilung el al 2005) Thus il
reqlllres salt to survive and appears in high er concentrations during the warmer summer
months
The optimum temperature fo r the grow th of V parahaemolylicus is 37 degC and at Ihis
optimum temperature the bac terium has generation times of8 to 9 minutes in water and 12 [ 0
18 minutes in seafood (Montville and Matthews 2008) Thi s bacterium is the most IKeiy
Vibrio species to be imp licated in food borne diseases although both V vulnificus and V
cholera may also causes food bom e infections (Lawley e l al 2008) Clinical isolates of V
parrhaemolyticus produce beta type hemolysis on blood aga r (Wagatsuma agar) The
hemolysis is ca ll ed the Kanagawa-phenomenon (KP) which is associated with the production
ofa thermo stabl e d irect hemolys inTOH (Boyd 2008) TOH caused damage to the euka ryotic
cells by acting as a pore fonning toxin that alters the ion balance of cells (Boyd 2008)
Figure 21 SEM observa tlOos of V plrh cm)JJ(cu~
Image source Belas amp Colwe ll ( 1982)
6
-- UI
23 Fresh vegetables
Consumpti on of raw vegetables is common throughout the world not onl y owing to Ihe health
benefit but they are thought to be free from disease causing pathogens However recent food
borne outbreaks worldwi de is associ ated to consumption of fresh frui ts uncooked vegetables
and unpasteurized juices (Gorny 2006) Fruits and vegetable especially those that are eaten
raw without peeling can be the vehicles for transmission of a wide range of mi croorganis m
(Erdogru l and Sener 2005)
Based on Haeton and Jones (2008) E coli Salmonella spp and Listeria spp are
commonly present in vegetable samples Meldrum el 01 (2009) report ed that the two recent
large ou tbreaks in the United Kingdom showed signi fi cant health problems that cou ld ari se
from consumption of contaminated salad Another study of the preva lence of pathogens in
vegetables was reported by Little el 01 (2007) in which they found Listeria monocylOgenes in
ready-to-eat mixed salads in the United Kingdom Several other studi es on d ifferent
microorga nism isolated from vegetabl es were also report ed recent ly by local researchers
(Chai el 01 2008 Han el 01 2009 Ponni ah el 0 2009)
In Mala ys ia the presence of V parahoemo yticus has also been repOIt ed in raw
vegetables samples from wet markets and supeLl11arkets in Selangor (Tunung et 0 20 I 0)
This same study discovered that raw vegetabl es such as OenGnthe st l7i(era (Japanese
parsley) and C[cumis sali vus (Cucumber) were contaminated with virulent V
7
parahaemoiYlicus Consequen tly these vegetables could act as a transmission route and
poses risk to consumers after consumption Tunung el 01 (2011) had also demonstrated that
the vegetable samples fro m wet market contained higher levels of V paraiJaemolylicls as
compared w ith supennarkets The research suggested that there are many factors that can
contri bu te to the distribution of V parahaemoiylicus such as temperature holding ti me as
well as hygiene and sanitation practice (Tunung el aI 2011) However there is no known
documented research on V parahaemolYlicus in raw vegetables in Sarawak
24 Clinical characteristic of V parahaelllolyliclis infections
II parahaemoiyticus causes three distinct illness syndromes which are gastroenteritis wound
or soft tissues infections and septicemia (Sillljee 2007) The most freq uen t syndrome is
gastroeuteritis The symptoms include dianhea with abdomina l cramps nausea vo miting
headache and low-grade fever (S tarlin 2005 WilU1 and Konema n 2006 Montville and
Matthew 2008) Sometimes the diarrhea is bloody w ith stools described as meat washed
since tl1e stool is reddish and watery (Qadri el aI 2009)
As for wound or tis sue in fections this coudition occurs by the exposure of broken skin
or wound to warm seawater and seafood dripp ings (Ralph and Currie 2007 Simjee 2007)
In severe cases serious wound infection will resu lt in necrotizing fasc iiti s (Ralph and Curri e
2007) Although less common in V paraiwemolyliclIs infection septicemia is characteri7cd
by fever or hypotension with the isolation of V parahaemolyticus in the blood Septicemia
typicall y occurs in person with underlying health problems such as liver disease diabetes
cancer or other immune disorder (Simjee 2007) V parahaemoyticlis can even ca use fala l
septicemia in an immune-compromised hosts
The symptoms usuall y occur on the average of 15 hours but can began as early as 4
hours and as late as 36 hours after exposure and this will continue up to 3 days (Barceloux
200S) However these illnesses are usua lly categorized as a short illnesses and no prolonged
enect will take place after recovery (Wright et al 2009)
25 Isolation of V parahael1lolyliclIs
For the isolation of V parahaemoyticus an initial emichment using alka line peptone wat er
(APW) with pH S5 would be useful as this medium supports the growth uf viable ce lls
(Simjee 2007) Plati ng of emichment broth onto TCBS agar has been widely used for
selective isolation of V parahaemolytics from foods (Thompson el ai 2006 Oi Pinto et al
201 0 Canizalez -Roman et ai 2011 ) An average of 24 hours incubation at 37 degC is requ ired
fo r typica l V parahaemolylicus to develop colonies that are smooth gree n (s u~rose nega tive)
and 2 to 3 nun in di ameter on TCBS (ISO 2007) However V parahaenlOlytlcus co lonies on
TCBS agar are difficult to distinguish vi suall y from other bacterial colonies since they can be
covered by yellow color produced by sucrose-fennenting bacteria (Thompson el 01 2006
Rambach 2007)
9
_ ------~-- i ~~
Another widely used selective medium is CHROMagar VibrioT On this medium V
pllrahaemoiy liclis will developed mau ve color colonies (Kudo et aI 2001 Oi Pi nto 2010
Canizalez-Roman 2011) This agar medium contains substrates for beta-galactosidase which
was developed specifically to differentiate V parahaemolytics from ot her bacteria by us ing a
chromogenic su bstrate instead of sugar fermentation in traditional growth med ia such as
TCBS (Hara-Kudo et 01 2001) Therefore it is more sensitiv e and accurate for identi fying V
porahllemolyticlIs in food samples rather than the use of Te ES (Oi Pinto 2010 Canizalez-
Roman 20 II)
26 Identification through Gram-stain and biochemical tests
V parahaemoiyticus is a Gram-negative bacterium th us this organi sm stained pink or red
after Gram staining (HPA 20073) Under microscopic observation a typical V
pllrahaemolyticus appeared as rod characterist ica lly curved or comma-shoped The bacterium
is also positi ve for motility under microscopic observations (ISO 2007 BPA 2007a)
V parahaemolylieus ca n be identified through a seri es of biochemical tests According
to Khan et 01 (2007) V para haemolyticus can be distinct ively differentia ted fro m the other
Vibrio spp by performing sa lt tolerance and acid fro m femlen tation tests A typical V
parahaemoiYlicus co lonies would not grow in AFW without th e presence of at least 3 of sali
10
~-------
but it can only endure the presence of salt up to 8 (ISO 2007 Khan el aI 2007) In the
other hand the fermentation test for acid production is the most important test to differentiate
between V parahaemolyliclIs and V vulnificus species (Khan el aI 2007) Jayasinge et ai
(2010) even suggested that the combination of salt toleranc e test Voges-Proskauer test and
ONPG test can provide a strai ghtforward differentiation of V parahaemoly liclis from the
other Vibrionaceae species (Figure 31)
Citrate test screens the ab ilit y of bacterial isolates to utilize ci trate as its carbon and
energy source (MacFaddin 2000) This test is a very important test to identify gram-negative
pathogens (Tang el aI 1998) The MR-VP medium which was used for Met hyl red and
Voges Proskauer test contains glucose and peptone Methyl red test screens for the bacterial
metaboli sm pathways for glucose fermentation while the Voges-Proskauer tes t is an assay for
organisms which ferment glucose with only one fermentation product usuall y acetic acid
The TS[ test is another useful test tha t can distinguish V parahaemolyticus from other
bacteria (ISO 2007) The TSJ agar med ium contains peptone fenous sulfate lactose sucrose
and a small amount of glucose (dex trose) The pH indi cator is phenol red This medium
differentiates bacteria based on their ability to fennent glucose lactose ando r sucrose and on
their abi lity to reduce sulfur to hydrogen sulfide (H S) The T SJ test also haj the abi lity to
determine the production of H]S (Kim et aI 1999)
In additi on other bi ochemica l tests such as oxidase lysine decarboxylase om ithi ne
decarboxylase ca talase and indole can also be used for the purpose of identifying V
II
-- ---- - ----------- - ~i
parahaemoyticus iso lates (ISO 2007 Jayasinghe et ai 2007 Khan et aI 2007) The
biochemical reactions o f V parahaemoiyticus are summarized in Table 21
Table 21 Siandard biochemical tesl reactions of V porahaemoyticus
TeSi S Reaction of V parahaem olyaclIs Refe rences Salt tol ance
+
Sali ne TSI agar KIA
No HS and gas production
Simmons Citrnh
Voges-Proskauer
Methyl-Red +
[SO (2007)
Jayasinge et al (2007)
Khan et of (2007)
ISO (2007)
Kim et I (1999)
Abide et til (1999)
Kim 11111 (1999)
Khan et til (2007)
Jayasinge el III (20 (0)
Kim et 0 (2 007)
Note (-) ~ nega live (+) ~ positi ve K ~ alka line ~ aC idic
12
CHAPT ER 3
MATERIALS AND METI IODS
31 Samples collection
Eighty raw vegetable samples were collected week ly from two wet markets (WI amp W2) and
two supennarkets (S 1 amp S2) in Kuching Sarawa k The collected samples consisted o f four
di fferent groups of vegetables which are regularly consumed ra w by the locals These samples
wcr~ rand omly selected within ten weeks -period which is from October 20 10 to January
20 11 Duri ng collection each sample was transferred to clean plastic bags labeled acco rding
to the place they were collected and were analyzed immed iately on arriva l to the labo rat ory
The types of raw vegetable sa mples obtained were as shown in Table 31
Tab le 31 Types o f raw vegetable samples co llected from wet markes and supcmlarkels
Group Loca l nam e English nam e Scienti fi c Home l mple coding No or samples
Soil Kubis Cabbage Brassica v eracea K 20 Roots Lobak Canmiddotot Douclfs carola C ~O
mera h Beans KaCtlO g Four-winged PsophOCaJjJ lls KB cO
bow l bean JeIlagulIolobus Shoot Tauge Bean-I_prout Vigna rad mc T 10 Total 80
13
---------~ I I
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
--
C
APW
g
KJA
NK
1111
mm
MR
NaCI
PCR
Spp
TeBS
TSI
VP
V parahaemoLytiCLIs Vp
WHO
LIST OF ABBREVIATIONS
percentage
degree celcius
Alkaline Peptone Water
gram
hyd rogen sulfite
alkaline slant acidic bUlt
acidic slant alkaline butt
microliter
milli liter
milimeter
Methyl-red
Sodium chloride
Polymerase Chain Reaction
species
Tiosulphate Citrate Bile Salt Sucrose
Tliple Sugar Iron
V oges-Proskaeur
Vibrio parallOemolyticlIs
World Heal th Organizati ons
v
~-
LIST OF TABLES
Tables Page
Table 21 Standard biochemical test reactio ns of V parahaemolyliclIs 12
Table 31 Types of raw vegetable samples collected from wet markets and 13
supermarkets
Table 41 Results for the presence of V parahaemolyticus on TCBS and 2 1
CHROMa gar Vibrio from the vegetable sa mples
Table 42 Result s of microscopic observations of the suspected V 27
parahaemolylicus iso lates
Table 43 Overall resuit of biochemical tests 24
vi
--bull i~
LIST OF FIG URES
Figures Page
Figurc 2t SEM observation o f Ii parahaemolyticus Image source Belas amp 6
Colwell (1 982)
and C I-IR OM agar Vib rio trade based on different sam pling locr io ns
Figure 31 Biochemical keys fo r the identification o f Vibrio species 17
Figure 41 The isolation o f Ii parQhaemolyticus on TCBS 22
Figure 42 Appearance of mixed colonies on CHROM agar Vib riotrade 22
Figure 43 Pure mauve co lonies obtained a fter sub -culturing 23
figurc 44 Reac tio ns of (a) B7KBS and (b) B8CW towards Voges -Proskauer test 24
Figure 45 Reactio ns of (a) B7KBS and (b) BI CW towards M dhly-red test 25
Figure 46 The reaction of a ll isolates towards citrate test 25
Figure 47 The reaction o f (a) B7KBS = KJA and (b) B3KW = AK fo r TSltes t 26
Figure 48 The reactio n o fB7KBS towards (a) 0 NaC I and (b) 8 Nae 27
Figu re 5 1 Bar graph o f the di stribution of presumptive pas iti ve sampl es on TCBS 30
Figure 52 Pie chart o f the distribu tion of presumptive Ii paraliaemolyliclls 31
between different vegetable types on CHROM aga r Vibrio
Figure 53 Pi e chart of the distribution of presumptive Ii parahaemolylicus 33
between di fferent vegetab le types on CHROMagar Vibriotrade
VII
--~bull i lj
Tbe Incidence of Vibrio p arahaellloly ticlis in Raw Vegetables at Local Wet Markets and Supermarkets in Kuching Sarawak
Sabatini lihob
Resource BIOIClhnoltlg) Progrtlm me Facu Ity or Resource Science Bud T~lln(llogy
Uni versity Malaysb Sa rawa k
ABSTRACT
Vibrio parawemoyliCLls is a foodbome pa thogen that is regular ly assoc iated with the consu mption o f
raw or undercooked seafood The mos t frequent sympto m of V parahaemoylicus infecr ion is
gastroenteritis Recen tly this pa thogen was also disco vered in raw vegetable samples in Peninsular
Malaysia Thus the aim of this study is to isolate and de tect V paahaemoylicu~ in raw vegdables T he samples which consisted of carrot fourmiddot winged bean cabbage and bea n sp ro ul were purc hased
tiom two wet markets and two supenn arkets in Kucll ing Sarawak In th is stud y alkaline peplone
water (A PW) wa s used as the enridunent medium The combinati o n o f two types o f selective agar
TCBS and CHROMa gar Vibrio nbull1 was successfully app lied to isolate the bacteria A fte r a series of
biochemical tests only 2 out of the 80 samples were pos itive for the p resence of V parahaemoylicLls
Although the peroentage of V pOrehaemolylics prevalence was low (2 5) the resu lts h ighli ghted
the fac t that raw vege tables could also be contaminated w It h V parahaenro~Vlicus
Key words Vib rio parahemotyfic~ raw vegetables TC BS and CHROMagar Vibrio rM
ABSTRAK
Vibrio parawcmoylicus merupakan sejenis patogen bmvaan makanafl )long sering dikairkan dengan
pengambiall makonon laul seewa men fa h Tonda paling kemp bagi j angkitall k parahwnJ r lvticlfs
ialah gasroenlritis TerbolU pafogen ini Ina dijumpa i di dalam sUlllp~1 scJyuran melJfah di
Semenanjung A1nlaYiia Kajial1 ini berlujuan un uk mengasmg dan mengesan V nanlhaemolliqi di
daall1 sampel $oyuran menfah Sampel yang lerdiri doripada obok merah kac(mg botol kobis dan
(l uge teah dlbeli dari dU(J pasai basah Clnd clua pasar raya di Kuchi lg Sorm lak Il l pepton beralktrli
(A PW) fel(lh digul1akan Ill1tuk memperbonyakko1 jwn Jah bakfcna Kombilw si pelggunaon agar
terpilih Tess dan CHROkiagar Vibrio T1 tdoh bejaya mel11ellcilkon ba( ur ia yallg dikehendoki
Selelah beberapa siri lljion biokimia didapafi 2 daripada 80 sampel relah dlcemQj oleh 1 rar(fhoenlJliJji(u~ Mcsklpun perafus kehadiran V norahaemolvticus adalah remJah (25~ o)
kepuflfsan kajian ini menunjukkan bahawo saYllran mental boleh dicemari ole Y paraha~moh IIcm
Kata klllt ci Vibrio oorqhaemol liclfs sayuran men fah TCBS dOll CHROtviagar Vihriotrade
-
CHAPTER 1
INTRODUCTION
11 Introduction
Foodbome illness assoc iated with consumption of contam inated food is a corrunon hea lth
problem that occurs worldwide The global inc idence of food bo rne ill ness is d ifficu lt to
estimate but it has been reported that in 2005 alone around 76 million cases of food bo rne
diseases resu lting in 325000 hospita liza tions and 5000 dea ths are es timated to occur in the
United State (WHO 20 11 ) In addition food contaminat ion also causes enormous socia l and
economical burden on the community A consumer research repon released on 2010 revea led
that the healt h-related costs of food-borne illnesses had cost up to 152 billion do llar a year
includ ing the costs of med ica l bills lost wages and lost produc tivi ty (Smii middot 2010) Thus
food safety is cun-entl y an increasing important pub lic health issue laquougovenunents all over
the world are intensify ing the ir efforts to improve food safety
Among the majo r foodbome illness caused by mi croorgan ism are Salmonellosis
Campylobacteriosis Listeriosis and infectio ns due to entero haemonha gic (caJing intes tinal
bleeding) E coli (WHO 2007) III recent years Vibriosis had emerged to be an impOItant
bac te rial disease that canses human intestinal and ext ra-intestinal infections a ll over the world
(Chan elol 2002 Garc ia el al 2009 and Harth et al 2009) The three Vibrio species o f
primary significance in foodbo rne illnesses are V cholerae (serogroups 01 non-Ol and
2
- bull i
0 139) V vulnijiclIs and V paralwemolyricus Presently gast roenteitis caused by V
parahaemolyticus is extensively documented due to its increasing outbreaks (Alam el 01
2003 Gwendelynne el 01 2005 Gorny 2006 Harth et 01 2009 Roque et 01 2009)
V parahaemolyriclls is widely present in brackish and marine waters and infection to
human is regula rl y associated with the consumption of contaminated raw or undercooked
scafood (Guoxia ng el 01 2009) Ea rli er studies also demonstrated that V parahoemolylicus
can be easily iso lated from costal water sediments suspended particles and plankton due to
its halophi lic characteristic (Marlina el 01 2007 Zinuneman el 01 2007 Zu lkifli el al
2009 Vimala et 01 2010) However recent foodbome outbreaks throughout the world have
been intensive ly linked to consu mption of fresh fruits vegetables and unpasteurized juices
(Gorny 2006) Most recently Tunu ng et 01 (20 10) had rep0l1ed th at the vegetable samples
from supermarkets and wet markets in Selangor Malaysia were posit ive for pathogeni c
species of V parahaemolylicls Conversely there is no ot her known documented research on
the prevalence of this species in vegetables
In this st udy vegetables from two wet markets and two supem1arkets in Kuching
Sa rawak were sampled for the presence of V parallOemolyliclis The sa moles underwent
selective enrichment with alkaline peptone water (APW) supp lemented with 3 NaCI Then
presumptive V parahaemolYlicus were iso lated using TeES agar and CH ROMagar VibrioT
Following isolat ion gram stai ning together with a series of biochemica l tests were perfonned
to assist in the identification and confirmation of the iso lates
3
- bull i ~
IZ Objectives
This study was underta ken with the fo llowing objecti ves
I To isolate presumptive V parahaemolYlicus from marketed vegetabl es samples
in Kuching Sarawak by using Te BS and CHROMagar VibrioTM
II To confirm th e suspected V parahaemolyficus iso lates from marketed
vegetables samples in Kuching Sarawak by usi ng Gram stain and biochemical
tests
4
- bull i ~
USI KhJtlmal Makumat U umk VMVl~m ItLAYSlA AAAWAK
CHAPTER 2
LITERATURE REVIEW
21 The genus Vibrio
The genus Vibrio belongs to the fami ly Vibrionaceae and it consists of at least 34 recogn ized
species (HPA 2007a) In general Vibrio spp are Gram-negat ive fac ulta tively anaerobic
mOlile curved rods bacteria with a single polar flagellum (FEHD 2005) In surface waters of
the world Vibrios are among the mos t common surface organisms (Adeleye et aI 20 J 0)
They are present in both marine and fresh wa ter habiwts and commonly associa ted with
aquatic animals Vibrio pp such as V choerae V parahaemoylicus V vu nificus V
aginoylieus V mimicus V luvia lis V filmissii V melselmikovii V tollisae and V
dalltsea are common human pathogens (Tison 1999 Fi nkelstein 2000) T hey account for a
significant proportion of human infections as most of these vibrios secrete enterotoxins to
foods water or in the gastrointestinal tract (Nishibuchi and DePao la 2005)
22 Vibrio paralJlell1oytictls
Vibrio parahaemoy licus which is formerly known as Pasteurella parahaeliloytlca is a
non-sporing Gram-negat ive facu ltatively-anaerobic bacterium (Curti s el 01 2003) This
5
- ~
halophilic bacterium inhabits warm estuarine waters world wide (Bilung el al 2005) Thus il
reqlllres salt to survive and appears in high er concentrations during the warmer summer
months
The optimum temperature fo r the grow th of V parahaemolylicus is 37 degC and at Ihis
optimum temperature the bac terium has generation times of8 to 9 minutes in water and 12 [ 0
18 minutes in seafood (Montville and Matthews 2008) Thi s bacterium is the most IKeiy
Vibrio species to be imp licated in food borne diseases although both V vulnificus and V
cholera may also causes food bom e infections (Lawley e l al 2008) Clinical isolates of V
parrhaemolyticus produce beta type hemolysis on blood aga r (Wagatsuma agar) The
hemolysis is ca ll ed the Kanagawa-phenomenon (KP) which is associated with the production
ofa thermo stabl e d irect hemolys inTOH (Boyd 2008) TOH caused damage to the euka ryotic
cells by acting as a pore fonning toxin that alters the ion balance of cells (Boyd 2008)
Figure 21 SEM observa tlOos of V plrh cm)JJ(cu~
Image source Belas amp Colwe ll ( 1982)
6
-- UI
23 Fresh vegetables
Consumpti on of raw vegetables is common throughout the world not onl y owing to Ihe health
benefit but they are thought to be free from disease causing pathogens However recent food
borne outbreaks worldwi de is associ ated to consumption of fresh frui ts uncooked vegetables
and unpasteurized juices (Gorny 2006) Fruits and vegetable especially those that are eaten
raw without peeling can be the vehicles for transmission of a wide range of mi croorganis m
(Erdogru l and Sener 2005)
Based on Haeton and Jones (2008) E coli Salmonella spp and Listeria spp are
commonly present in vegetable samples Meldrum el 01 (2009) report ed that the two recent
large ou tbreaks in the United Kingdom showed signi fi cant health problems that cou ld ari se
from consumption of contaminated salad Another study of the preva lence of pathogens in
vegetables was reported by Little el 01 (2007) in which they found Listeria monocylOgenes in
ready-to-eat mixed salads in the United Kingdom Several other studi es on d ifferent
microorga nism isolated from vegetabl es were also report ed recent ly by local researchers
(Chai el 01 2008 Han el 01 2009 Ponni ah el 0 2009)
In Mala ys ia the presence of V parahoemo yticus has also been repOIt ed in raw
vegetables samples from wet markets and supeLl11arkets in Selangor (Tunung et 0 20 I 0)
This same study discovered that raw vegetabl es such as OenGnthe st l7i(era (Japanese
parsley) and C[cumis sali vus (Cucumber) were contaminated with virulent V
7
parahaemoiYlicus Consequen tly these vegetables could act as a transmission route and
poses risk to consumers after consumption Tunung el 01 (2011) had also demonstrated that
the vegetable samples fro m wet market contained higher levels of V paraiJaemolylicls as
compared w ith supennarkets The research suggested that there are many factors that can
contri bu te to the distribution of V parahaemoiylicus such as temperature holding ti me as
well as hygiene and sanitation practice (Tunung el aI 2011) However there is no known
documented research on V parahaemolYlicus in raw vegetables in Sarawak
24 Clinical characteristic of V parahaelllolyliclis infections
II parahaemoiyticus causes three distinct illness syndromes which are gastroenteritis wound
or soft tissues infections and septicemia (Sillljee 2007) The most freq uen t syndrome is
gastroeuteritis The symptoms include dianhea with abdomina l cramps nausea vo miting
headache and low-grade fever (S tarlin 2005 WilU1 and Konema n 2006 Montville and
Matthew 2008) Sometimes the diarrhea is bloody w ith stools described as meat washed
since tl1e stool is reddish and watery (Qadri el aI 2009)
As for wound or tis sue in fections this coudition occurs by the exposure of broken skin
or wound to warm seawater and seafood dripp ings (Ralph and Currie 2007 Simjee 2007)
In severe cases serious wound infection will resu lt in necrotizing fasc iiti s (Ralph and Curri e
2007) Although less common in V paraiwemolyliclIs infection septicemia is characteri7cd
by fever or hypotension with the isolation of V parahaemolyticus in the blood Septicemia
typicall y occurs in person with underlying health problems such as liver disease diabetes
cancer or other immune disorder (Simjee 2007) V parahaemoyticlis can even ca use fala l
septicemia in an immune-compromised hosts
The symptoms usuall y occur on the average of 15 hours but can began as early as 4
hours and as late as 36 hours after exposure and this will continue up to 3 days (Barceloux
200S) However these illnesses are usua lly categorized as a short illnesses and no prolonged
enect will take place after recovery (Wright et al 2009)
25 Isolation of V parahael1lolyliclIs
For the isolation of V parahaemoyticus an initial emichment using alka line peptone wat er
(APW) with pH S5 would be useful as this medium supports the growth uf viable ce lls
(Simjee 2007) Plati ng of emichment broth onto TCBS agar has been widely used for
selective isolation of V parahaemolytics from foods (Thompson el ai 2006 Oi Pinto et al
201 0 Canizalez -Roman et ai 2011 ) An average of 24 hours incubation at 37 degC is requ ired
fo r typica l V parahaemolylicus to develop colonies that are smooth gree n (s u~rose nega tive)
and 2 to 3 nun in di ameter on TCBS (ISO 2007) However V parahaenlOlytlcus co lonies on
TCBS agar are difficult to distinguish vi suall y from other bacterial colonies since they can be
covered by yellow color produced by sucrose-fennenting bacteria (Thompson el 01 2006
Rambach 2007)
9
_ ------~-- i ~~
Another widely used selective medium is CHROMagar VibrioT On this medium V
pllrahaemoiy liclis will developed mau ve color colonies (Kudo et aI 2001 Oi Pi nto 2010
Canizalez-Roman 2011) This agar medium contains substrates for beta-galactosidase which
was developed specifically to differentiate V parahaemolytics from ot her bacteria by us ing a
chromogenic su bstrate instead of sugar fermentation in traditional growth med ia such as
TCBS (Hara-Kudo et 01 2001) Therefore it is more sensitiv e and accurate for identi fying V
porahllemolyticlIs in food samples rather than the use of Te ES (Oi Pinto 2010 Canizalez-
Roman 20 II)
26 Identification through Gram-stain and biochemical tests
V parahaemoiyticus is a Gram-negative bacterium th us this organi sm stained pink or red
after Gram staining (HPA 20073) Under microscopic observation a typical V
pllrahaemolyticus appeared as rod characterist ica lly curved or comma-shoped The bacterium
is also positi ve for motility under microscopic observations (ISO 2007 BPA 2007a)
V parahaemolylieus ca n be identified through a seri es of biochemical tests According
to Khan et 01 (2007) V para haemolyticus can be distinct ively differentia ted fro m the other
Vibrio spp by performing sa lt tolerance and acid fro m femlen tation tests A typical V
parahaemoiYlicus co lonies would not grow in AFW without th e presence of at least 3 of sali
10
~-------
but it can only endure the presence of salt up to 8 (ISO 2007 Khan el aI 2007) In the
other hand the fermentation test for acid production is the most important test to differentiate
between V parahaemolyliclIs and V vulnificus species (Khan el aI 2007) Jayasinge et ai
(2010) even suggested that the combination of salt toleranc e test Voges-Proskauer test and
ONPG test can provide a strai ghtforward differentiation of V parahaemoly liclis from the
other Vibrionaceae species (Figure 31)
Citrate test screens the ab ilit y of bacterial isolates to utilize ci trate as its carbon and
energy source (MacFaddin 2000) This test is a very important test to identify gram-negative
pathogens (Tang el aI 1998) The MR-VP medium which was used for Met hyl red and
Voges Proskauer test contains glucose and peptone Methyl red test screens for the bacterial
metaboli sm pathways for glucose fermentation while the Voges-Proskauer tes t is an assay for
organisms which ferment glucose with only one fermentation product usuall y acetic acid
The TS[ test is another useful test tha t can distinguish V parahaemolyticus from other
bacteria (ISO 2007) The TSJ agar med ium contains peptone fenous sulfate lactose sucrose
and a small amount of glucose (dex trose) The pH indi cator is phenol red This medium
differentiates bacteria based on their ability to fennent glucose lactose ando r sucrose and on
their abi lity to reduce sulfur to hydrogen sulfide (H S) The T SJ test also haj the abi lity to
determine the production of H]S (Kim et aI 1999)
In additi on other bi ochemica l tests such as oxidase lysine decarboxylase om ithi ne
decarboxylase ca talase and indole can also be used for the purpose of identifying V
II
-- ---- - ----------- - ~i
parahaemoyticus iso lates (ISO 2007 Jayasinghe et ai 2007 Khan et aI 2007) The
biochemical reactions o f V parahaemoiyticus are summarized in Table 21
Table 21 Siandard biochemical tesl reactions of V porahaemoyticus
TeSi S Reaction of V parahaem olyaclIs Refe rences Salt tol ance
+
Sali ne TSI agar KIA
No HS and gas production
Simmons Citrnh
Voges-Proskauer
Methyl-Red +
[SO (2007)
Jayasinge et al (2007)
Khan et of (2007)
ISO (2007)
Kim et I (1999)
Abide et til (1999)
Kim 11111 (1999)
Khan et til (2007)
Jayasinge el III (20 (0)
Kim et 0 (2 007)
Note (-) ~ nega live (+) ~ positi ve K ~ alka line ~ aC idic
12
CHAPT ER 3
MATERIALS AND METI IODS
31 Samples collection
Eighty raw vegetable samples were collected week ly from two wet markets (WI amp W2) and
two supennarkets (S 1 amp S2) in Kuching Sarawa k The collected samples consisted o f four
di fferent groups of vegetables which are regularly consumed ra w by the locals These samples
wcr~ rand omly selected within ten weeks -period which is from October 20 10 to January
20 11 Duri ng collection each sample was transferred to clean plastic bags labeled acco rding
to the place they were collected and were analyzed immed iately on arriva l to the labo rat ory
The types of raw vegetable sa mples obtained were as shown in Table 31
Tab le 31 Types o f raw vegetable samples co llected from wet markes and supcmlarkels
Group Loca l nam e English nam e Scienti fi c Home l mple coding No or samples
Soil Kubis Cabbage Brassica v eracea K 20 Roots Lobak Canmiddotot Douclfs carola C ~O
mera h Beans KaCtlO g Four-winged PsophOCaJjJ lls KB cO
bow l bean JeIlagulIolobus Shoot Tauge Bean-I_prout Vigna rad mc T 10 Total 80
13
---------~ I I
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
LIST OF TABLES
Tables Page
Table 21 Standard biochemical test reactio ns of V parahaemolyliclIs 12
Table 31 Types of raw vegetable samples collected from wet markets and 13
supermarkets
Table 41 Results for the presence of V parahaemolyticus on TCBS and 2 1
CHROMa gar Vibrio from the vegetable sa mples
Table 42 Result s of microscopic observations of the suspected V 27
parahaemolylicus iso lates
Table 43 Overall resuit of biochemical tests 24
vi
--bull i~
LIST OF FIG URES
Figures Page
Figurc 2t SEM observation o f Ii parahaemolyticus Image source Belas amp 6
Colwell (1 982)
and C I-IR OM agar Vib rio trade based on different sam pling locr io ns
Figure 31 Biochemical keys fo r the identification o f Vibrio species 17
Figure 41 The isolation o f Ii parQhaemolyticus on TCBS 22
Figure 42 Appearance of mixed colonies on CHROM agar Vib riotrade 22
Figure 43 Pure mauve co lonies obtained a fter sub -culturing 23
figurc 44 Reac tio ns of (a) B7KBS and (b) B8CW towards Voges -Proskauer test 24
Figure 45 Reactio ns of (a) B7KBS and (b) BI CW towards M dhly-red test 25
Figure 46 The reaction of a ll isolates towards citrate test 25
Figure 47 The reaction o f (a) B7KBS = KJA and (b) B3KW = AK fo r TSltes t 26
Figure 48 The reactio n o fB7KBS towards (a) 0 NaC I and (b) 8 Nae 27
Figu re 5 1 Bar graph o f the di stribution of presumptive pas iti ve sampl es on TCBS 30
Figure 52 Pie chart o f the distribu tion of presumptive Ii paraliaemolyliclls 31
between different vegetable types on CHROM aga r Vibrio
Figure 53 Pi e chart of the distribution of presumptive Ii parahaemolylicus 33
between di fferent vegetab le types on CHROMagar Vibriotrade
VII
--~bull i lj
Tbe Incidence of Vibrio p arahaellloly ticlis in Raw Vegetables at Local Wet Markets and Supermarkets in Kuching Sarawak
Sabatini lihob
Resource BIOIClhnoltlg) Progrtlm me Facu Ity or Resource Science Bud T~lln(llogy
Uni versity Malaysb Sa rawa k
ABSTRACT
Vibrio parawemoyliCLls is a foodbome pa thogen that is regular ly assoc iated with the consu mption o f
raw or undercooked seafood The mos t frequent sympto m of V parahaemoylicus infecr ion is
gastroenteritis Recen tly this pa thogen was also disco vered in raw vegetable samples in Peninsular
Malaysia Thus the aim of this study is to isolate and de tect V paahaemoylicu~ in raw vegdables T he samples which consisted of carrot fourmiddot winged bean cabbage and bea n sp ro ul were purc hased
tiom two wet markets and two supenn arkets in Kucll ing Sarawak In th is stud y alkaline peplone
water (A PW) wa s used as the enridunent medium The combinati o n o f two types o f selective agar
TCBS and CHROMa gar Vibrio nbull1 was successfully app lied to isolate the bacteria A fte r a series of
biochemical tests only 2 out of the 80 samples were pos itive for the p resence of V parahaemoylicLls
Although the peroentage of V pOrehaemolylics prevalence was low (2 5) the resu lts h ighli ghted
the fac t that raw vege tables could also be contaminated w It h V parahaenro~Vlicus
Key words Vib rio parahemotyfic~ raw vegetables TC BS and CHROMagar Vibrio rM
ABSTRAK
Vibrio parawcmoylicus merupakan sejenis patogen bmvaan makanafl )long sering dikairkan dengan
pengambiall makonon laul seewa men fa h Tonda paling kemp bagi j angkitall k parahwnJ r lvticlfs
ialah gasroenlritis TerbolU pafogen ini Ina dijumpa i di dalam sUlllp~1 scJyuran melJfah di
Semenanjung A1nlaYiia Kajial1 ini berlujuan un uk mengasmg dan mengesan V nanlhaemolliqi di
daall1 sampel $oyuran menfah Sampel yang lerdiri doripada obok merah kac(mg botol kobis dan
(l uge teah dlbeli dari dU(J pasai basah Clnd clua pasar raya di Kuchi lg Sorm lak Il l pepton beralktrli
(A PW) fel(lh digul1akan Ill1tuk memperbonyakko1 jwn Jah bakfcna Kombilw si pelggunaon agar
terpilih Tess dan CHROkiagar Vibrio T1 tdoh bejaya mel11ellcilkon ba( ur ia yallg dikehendoki
Selelah beberapa siri lljion biokimia didapafi 2 daripada 80 sampel relah dlcemQj oleh 1 rar(fhoenlJliJji(u~ Mcsklpun perafus kehadiran V norahaemolvticus adalah remJah (25~ o)
kepuflfsan kajian ini menunjukkan bahawo saYllran mental boleh dicemari ole Y paraha~moh IIcm
Kata klllt ci Vibrio oorqhaemol liclfs sayuran men fah TCBS dOll CHROtviagar Vihriotrade
-
CHAPTER 1
INTRODUCTION
11 Introduction
Foodbome illness assoc iated with consumption of contam inated food is a corrunon hea lth
problem that occurs worldwide The global inc idence of food bo rne ill ness is d ifficu lt to
estimate but it has been reported that in 2005 alone around 76 million cases of food bo rne
diseases resu lting in 325000 hospita liza tions and 5000 dea ths are es timated to occur in the
United State (WHO 20 11 ) In addition food contaminat ion also causes enormous socia l and
economical burden on the community A consumer research repon released on 2010 revea led
that the healt h-related costs of food-borne illnesses had cost up to 152 billion do llar a year
includ ing the costs of med ica l bills lost wages and lost produc tivi ty (Smii middot 2010) Thus
food safety is cun-entl y an increasing important pub lic health issue laquougovenunents all over
the world are intensify ing the ir efforts to improve food safety
Among the majo r foodbome illness caused by mi croorgan ism are Salmonellosis
Campylobacteriosis Listeriosis and infectio ns due to entero haemonha gic (caJing intes tinal
bleeding) E coli (WHO 2007) III recent years Vibriosis had emerged to be an impOItant
bac te rial disease that canses human intestinal and ext ra-intestinal infections a ll over the world
(Chan elol 2002 Garc ia el al 2009 and Harth et al 2009) The three Vibrio species o f
primary significance in foodbo rne illnesses are V cholerae (serogroups 01 non-Ol and
2
- bull i
0 139) V vulnijiclIs and V paralwemolyricus Presently gast roenteitis caused by V
parahaemolyticus is extensively documented due to its increasing outbreaks (Alam el 01
2003 Gwendelynne el 01 2005 Gorny 2006 Harth et 01 2009 Roque et 01 2009)
V parahaemolyriclls is widely present in brackish and marine waters and infection to
human is regula rl y associated with the consumption of contaminated raw or undercooked
scafood (Guoxia ng el 01 2009) Ea rli er studies also demonstrated that V parahoemolylicus
can be easily iso lated from costal water sediments suspended particles and plankton due to
its halophi lic characteristic (Marlina el 01 2007 Zinuneman el 01 2007 Zu lkifli el al
2009 Vimala et 01 2010) However recent foodbome outbreaks throughout the world have
been intensive ly linked to consu mption of fresh fruits vegetables and unpasteurized juices
(Gorny 2006) Most recently Tunu ng et 01 (20 10) had rep0l1ed th at the vegetable samples
from supermarkets and wet markets in Selangor Malaysia were posit ive for pathogeni c
species of V parahaemolylicls Conversely there is no ot her known documented research on
the prevalence of this species in vegetables
In this st udy vegetables from two wet markets and two supem1arkets in Kuching
Sa rawak were sampled for the presence of V parallOemolyliclis The sa moles underwent
selective enrichment with alkaline peptone water (APW) supp lemented with 3 NaCI Then
presumptive V parahaemolYlicus were iso lated using TeES agar and CH ROMagar VibrioT
Following isolat ion gram stai ning together with a series of biochemica l tests were perfonned
to assist in the identification and confirmation of the iso lates
3
- bull i ~
IZ Objectives
This study was underta ken with the fo llowing objecti ves
I To isolate presumptive V parahaemolYlicus from marketed vegetabl es samples
in Kuching Sarawak by using Te BS and CHROMagar VibrioTM
II To confirm th e suspected V parahaemolyficus iso lates from marketed
vegetables samples in Kuching Sarawak by usi ng Gram stain and biochemical
tests
4
- bull i ~
USI KhJtlmal Makumat U umk VMVl~m ItLAYSlA AAAWAK
CHAPTER 2
LITERATURE REVIEW
21 The genus Vibrio
The genus Vibrio belongs to the fami ly Vibrionaceae and it consists of at least 34 recogn ized
species (HPA 2007a) In general Vibrio spp are Gram-negat ive fac ulta tively anaerobic
mOlile curved rods bacteria with a single polar flagellum (FEHD 2005) In surface waters of
the world Vibrios are among the mos t common surface organisms (Adeleye et aI 20 J 0)
They are present in both marine and fresh wa ter habiwts and commonly associa ted with
aquatic animals Vibrio pp such as V choerae V parahaemoylicus V vu nificus V
aginoylieus V mimicus V luvia lis V filmissii V melselmikovii V tollisae and V
dalltsea are common human pathogens (Tison 1999 Fi nkelstein 2000) T hey account for a
significant proportion of human infections as most of these vibrios secrete enterotoxins to
foods water or in the gastrointestinal tract (Nishibuchi and DePao la 2005)
22 Vibrio paralJlell1oytictls
Vibrio parahaemoy licus which is formerly known as Pasteurella parahaeliloytlca is a
non-sporing Gram-negat ive facu ltatively-anaerobic bacterium (Curti s el 01 2003) This
5
- ~
halophilic bacterium inhabits warm estuarine waters world wide (Bilung el al 2005) Thus il
reqlllres salt to survive and appears in high er concentrations during the warmer summer
months
The optimum temperature fo r the grow th of V parahaemolylicus is 37 degC and at Ihis
optimum temperature the bac terium has generation times of8 to 9 minutes in water and 12 [ 0
18 minutes in seafood (Montville and Matthews 2008) Thi s bacterium is the most IKeiy
Vibrio species to be imp licated in food borne diseases although both V vulnificus and V
cholera may also causes food bom e infections (Lawley e l al 2008) Clinical isolates of V
parrhaemolyticus produce beta type hemolysis on blood aga r (Wagatsuma agar) The
hemolysis is ca ll ed the Kanagawa-phenomenon (KP) which is associated with the production
ofa thermo stabl e d irect hemolys inTOH (Boyd 2008) TOH caused damage to the euka ryotic
cells by acting as a pore fonning toxin that alters the ion balance of cells (Boyd 2008)
Figure 21 SEM observa tlOos of V plrh cm)JJ(cu~
Image source Belas amp Colwe ll ( 1982)
6
-- UI
23 Fresh vegetables
Consumpti on of raw vegetables is common throughout the world not onl y owing to Ihe health
benefit but they are thought to be free from disease causing pathogens However recent food
borne outbreaks worldwi de is associ ated to consumption of fresh frui ts uncooked vegetables
and unpasteurized juices (Gorny 2006) Fruits and vegetable especially those that are eaten
raw without peeling can be the vehicles for transmission of a wide range of mi croorganis m
(Erdogru l and Sener 2005)
Based on Haeton and Jones (2008) E coli Salmonella spp and Listeria spp are
commonly present in vegetable samples Meldrum el 01 (2009) report ed that the two recent
large ou tbreaks in the United Kingdom showed signi fi cant health problems that cou ld ari se
from consumption of contaminated salad Another study of the preva lence of pathogens in
vegetables was reported by Little el 01 (2007) in which they found Listeria monocylOgenes in
ready-to-eat mixed salads in the United Kingdom Several other studi es on d ifferent
microorga nism isolated from vegetabl es were also report ed recent ly by local researchers
(Chai el 01 2008 Han el 01 2009 Ponni ah el 0 2009)
In Mala ys ia the presence of V parahoemo yticus has also been repOIt ed in raw
vegetables samples from wet markets and supeLl11arkets in Selangor (Tunung et 0 20 I 0)
This same study discovered that raw vegetabl es such as OenGnthe st l7i(era (Japanese
parsley) and C[cumis sali vus (Cucumber) were contaminated with virulent V
7
parahaemoiYlicus Consequen tly these vegetables could act as a transmission route and
poses risk to consumers after consumption Tunung el 01 (2011) had also demonstrated that
the vegetable samples fro m wet market contained higher levels of V paraiJaemolylicls as
compared w ith supennarkets The research suggested that there are many factors that can
contri bu te to the distribution of V parahaemoiylicus such as temperature holding ti me as
well as hygiene and sanitation practice (Tunung el aI 2011) However there is no known
documented research on V parahaemolYlicus in raw vegetables in Sarawak
24 Clinical characteristic of V parahaelllolyliclis infections
II parahaemoiyticus causes three distinct illness syndromes which are gastroenteritis wound
or soft tissues infections and septicemia (Sillljee 2007) The most freq uen t syndrome is
gastroeuteritis The symptoms include dianhea with abdomina l cramps nausea vo miting
headache and low-grade fever (S tarlin 2005 WilU1 and Konema n 2006 Montville and
Matthew 2008) Sometimes the diarrhea is bloody w ith stools described as meat washed
since tl1e stool is reddish and watery (Qadri el aI 2009)
As for wound or tis sue in fections this coudition occurs by the exposure of broken skin
or wound to warm seawater and seafood dripp ings (Ralph and Currie 2007 Simjee 2007)
In severe cases serious wound infection will resu lt in necrotizing fasc iiti s (Ralph and Curri e
2007) Although less common in V paraiwemolyliclIs infection septicemia is characteri7cd
by fever or hypotension with the isolation of V parahaemolyticus in the blood Septicemia
typicall y occurs in person with underlying health problems such as liver disease diabetes
cancer or other immune disorder (Simjee 2007) V parahaemoyticlis can even ca use fala l
septicemia in an immune-compromised hosts
The symptoms usuall y occur on the average of 15 hours but can began as early as 4
hours and as late as 36 hours after exposure and this will continue up to 3 days (Barceloux
200S) However these illnesses are usua lly categorized as a short illnesses and no prolonged
enect will take place after recovery (Wright et al 2009)
25 Isolation of V parahael1lolyliclIs
For the isolation of V parahaemoyticus an initial emichment using alka line peptone wat er
(APW) with pH S5 would be useful as this medium supports the growth uf viable ce lls
(Simjee 2007) Plati ng of emichment broth onto TCBS agar has been widely used for
selective isolation of V parahaemolytics from foods (Thompson el ai 2006 Oi Pinto et al
201 0 Canizalez -Roman et ai 2011 ) An average of 24 hours incubation at 37 degC is requ ired
fo r typica l V parahaemolylicus to develop colonies that are smooth gree n (s u~rose nega tive)
and 2 to 3 nun in di ameter on TCBS (ISO 2007) However V parahaenlOlytlcus co lonies on
TCBS agar are difficult to distinguish vi suall y from other bacterial colonies since they can be
covered by yellow color produced by sucrose-fennenting bacteria (Thompson el 01 2006
Rambach 2007)
9
_ ------~-- i ~~
Another widely used selective medium is CHROMagar VibrioT On this medium V
pllrahaemoiy liclis will developed mau ve color colonies (Kudo et aI 2001 Oi Pi nto 2010
Canizalez-Roman 2011) This agar medium contains substrates for beta-galactosidase which
was developed specifically to differentiate V parahaemolytics from ot her bacteria by us ing a
chromogenic su bstrate instead of sugar fermentation in traditional growth med ia such as
TCBS (Hara-Kudo et 01 2001) Therefore it is more sensitiv e and accurate for identi fying V
porahllemolyticlIs in food samples rather than the use of Te ES (Oi Pinto 2010 Canizalez-
Roman 20 II)
26 Identification through Gram-stain and biochemical tests
V parahaemoiyticus is a Gram-negative bacterium th us this organi sm stained pink or red
after Gram staining (HPA 20073) Under microscopic observation a typical V
pllrahaemolyticus appeared as rod characterist ica lly curved or comma-shoped The bacterium
is also positi ve for motility under microscopic observations (ISO 2007 BPA 2007a)
V parahaemolylieus ca n be identified through a seri es of biochemical tests According
to Khan et 01 (2007) V para haemolyticus can be distinct ively differentia ted fro m the other
Vibrio spp by performing sa lt tolerance and acid fro m femlen tation tests A typical V
parahaemoiYlicus co lonies would not grow in AFW without th e presence of at least 3 of sali
10
~-------
but it can only endure the presence of salt up to 8 (ISO 2007 Khan el aI 2007) In the
other hand the fermentation test for acid production is the most important test to differentiate
between V parahaemolyliclIs and V vulnificus species (Khan el aI 2007) Jayasinge et ai
(2010) even suggested that the combination of salt toleranc e test Voges-Proskauer test and
ONPG test can provide a strai ghtforward differentiation of V parahaemoly liclis from the
other Vibrionaceae species (Figure 31)
Citrate test screens the ab ilit y of bacterial isolates to utilize ci trate as its carbon and
energy source (MacFaddin 2000) This test is a very important test to identify gram-negative
pathogens (Tang el aI 1998) The MR-VP medium which was used for Met hyl red and
Voges Proskauer test contains glucose and peptone Methyl red test screens for the bacterial
metaboli sm pathways for glucose fermentation while the Voges-Proskauer tes t is an assay for
organisms which ferment glucose with only one fermentation product usuall y acetic acid
The TS[ test is another useful test tha t can distinguish V parahaemolyticus from other
bacteria (ISO 2007) The TSJ agar med ium contains peptone fenous sulfate lactose sucrose
and a small amount of glucose (dex trose) The pH indi cator is phenol red This medium
differentiates bacteria based on their ability to fennent glucose lactose ando r sucrose and on
their abi lity to reduce sulfur to hydrogen sulfide (H S) The T SJ test also haj the abi lity to
determine the production of H]S (Kim et aI 1999)
In additi on other bi ochemica l tests such as oxidase lysine decarboxylase om ithi ne
decarboxylase ca talase and indole can also be used for the purpose of identifying V
II
-- ---- - ----------- - ~i
parahaemoyticus iso lates (ISO 2007 Jayasinghe et ai 2007 Khan et aI 2007) The
biochemical reactions o f V parahaemoiyticus are summarized in Table 21
Table 21 Siandard biochemical tesl reactions of V porahaemoyticus
TeSi S Reaction of V parahaem olyaclIs Refe rences Salt tol ance
+
Sali ne TSI agar KIA
No HS and gas production
Simmons Citrnh
Voges-Proskauer
Methyl-Red +
[SO (2007)
Jayasinge et al (2007)
Khan et of (2007)
ISO (2007)
Kim et I (1999)
Abide et til (1999)
Kim 11111 (1999)
Khan et til (2007)
Jayasinge el III (20 (0)
Kim et 0 (2 007)
Note (-) ~ nega live (+) ~ positi ve K ~ alka line ~ aC idic
12
CHAPT ER 3
MATERIALS AND METI IODS
31 Samples collection
Eighty raw vegetable samples were collected week ly from two wet markets (WI amp W2) and
two supennarkets (S 1 amp S2) in Kuching Sarawa k The collected samples consisted o f four
di fferent groups of vegetables which are regularly consumed ra w by the locals These samples
wcr~ rand omly selected within ten weeks -period which is from October 20 10 to January
20 11 Duri ng collection each sample was transferred to clean plastic bags labeled acco rding
to the place they were collected and were analyzed immed iately on arriva l to the labo rat ory
The types of raw vegetable sa mples obtained were as shown in Table 31
Tab le 31 Types o f raw vegetable samples co llected from wet markes and supcmlarkels
Group Loca l nam e English nam e Scienti fi c Home l mple coding No or samples
Soil Kubis Cabbage Brassica v eracea K 20 Roots Lobak Canmiddotot Douclfs carola C ~O
mera h Beans KaCtlO g Four-winged PsophOCaJjJ lls KB cO
bow l bean JeIlagulIolobus Shoot Tauge Bean-I_prout Vigna rad mc T 10 Total 80
13
---------~ I I
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
LIST OF FIG URES
Figures Page
Figurc 2t SEM observation o f Ii parahaemolyticus Image source Belas amp 6
Colwell (1 982)
and C I-IR OM agar Vib rio trade based on different sam pling locr io ns
Figure 31 Biochemical keys fo r the identification o f Vibrio species 17
Figure 41 The isolation o f Ii parQhaemolyticus on TCBS 22
Figure 42 Appearance of mixed colonies on CHROM agar Vib riotrade 22
Figure 43 Pure mauve co lonies obtained a fter sub -culturing 23
figurc 44 Reac tio ns of (a) B7KBS and (b) B8CW towards Voges -Proskauer test 24
Figure 45 Reactio ns of (a) B7KBS and (b) BI CW towards M dhly-red test 25
Figure 46 The reaction of a ll isolates towards citrate test 25
Figure 47 The reaction o f (a) B7KBS = KJA and (b) B3KW = AK fo r TSltes t 26
Figure 48 The reactio n o fB7KBS towards (a) 0 NaC I and (b) 8 Nae 27
Figu re 5 1 Bar graph o f the di stribution of presumptive pas iti ve sampl es on TCBS 30
Figure 52 Pie chart o f the distribu tion of presumptive Ii paraliaemolyliclls 31
between different vegetable types on CHROM aga r Vibrio
Figure 53 Pi e chart of the distribution of presumptive Ii parahaemolylicus 33
between di fferent vegetab le types on CHROMagar Vibriotrade
VII
--~bull i lj
Tbe Incidence of Vibrio p arahaellloly ticlis in Raw Vegetables at Local Wet Markets and Supermarkets in Kuching Sarawak
Sabatini lihob
Resource BIOIClhnoltlg) Progrtlm me Facu Ity or Resource Science Bud T~lln(llogy
Uni versity Malaysb Sa rawa k
ABSTRACT
Vibrio parawemoyliCLls is a foodbome pa thogen that is regular ly assoc iated with the consu mption o f
raw or undercooked seafood The mos t frequent sympto m of V parahaemoylicus infecr ion is
gastroenteritis Recen tly this pa thogen was also disco vered in raw vegetable samples in Peninsular
Malaysia Thus the aim of this study is to isolate and de tect V paahaemoylicu~ in raw vegdables T he samples which consisted of carrot fourmiddot winged bean cabbage and bea n sp ro ul were purc hased
tiom two wet markets and two supenn arkets in Kucll ing Sarawak In th is stud y alkaline peplone
water (A PW) wa s used as the enridunent medium The combinati o n o f two types o f selective agar
TCBS and CHROMa gar Vibrio nbull1 was successfully app lied to isolate the bacteria A fte r a series of
biochemical tests only 2 out of the 80 samples were pos itive for the p resence of V parahaemoylicLls
Although the peroentage of V pOrehaemolylics prevalence was low (2 5) the resu lts h ighli ghted
the fac t that raw vege tables could also be contaminated w It h V parahaenro~Vlicus
Key words Vib rio parahemotyfic~ raw vegetables TC BS and CHROMagar Vibrio rM
ABSTRAK
Vibrio parawcmoylicus merupakan sejenis patogen bmvaan makanafl )long sering dikairkan dengan
pengambiall makonon laul seewa men fa h Tonda paling kemp bagi j angkitall k parahwnJ r lvticlfs
ialah gasroenlritis TerbolU pafogen ini Ina dijumpa i di dalam sUlllp~1 scJyuran melJfah di
Semenanjung A1nlaYiia Kajial1 ini berlujuan un uk mengasmg dan mengesan V nanlhaemolliqi di
daall1 sampel $oyuran menfah Sampel yang lerdiri doripada obok merah kac(mg botol kobis dan
(l uge teah dlbeli dari dU(J pasai basah Clnd clua pasar raya di Kuchi lg Sorm lak Il l pepton beralktrli
(A PW) fel(lh digul1akan Ill1tuk memperbonyakko1 jwn Jah bakfcna Kombilw si pelggunaon agar
terpilih Tess dan CHROkiagar Vibrio T1 tdoh bejaya mel11ellcilkon ba( ur ia yallg dikehendoki
Selelah beberapa siri lljion biokimia didapafi 2 daripada 80 sampel relah dlcemQj oleh 1 rar(fhoenlJliJji(u~ Mcsklpun perafus kehadiran V norahaemolvticus adalah remJah (25~ o)
kepuflfsan kajian ini menunjukkan bahawo saYllran mental boleh dicemari ole Y paraha~moh IIcm
Kata klllt ci Vibrio oorqhaemol liclfs sayuran men fah TCBS dOll CHROtviagar Vihriotrade
-
CHAPTER 1
INTRODUCTION
11 Introduction
Foodbome illness assoc iated with consumption of contam inated food is a corrunon hea lth
problem that occurs worldwide The global inc idence of food bo rne ill ness is d ifficu lt to
estimate but it has been reported that in 2005 alone around 76 million cases of food bo rne
diseases resu lting in 325000 hospita liza tions and 5000 dea ths are es timated to occur in the
United State (WHO 20 11 ) In addition food contaminat ion also causes enormous socia l and
economical burden on the community A consumer research repon released on 2010 revea led
that the healt h-related costs of food-borne illnesses had cost up to 152 billion do llar a year
includ ing the costs of med ica l bills lost wages and lost produc tivi ty (Smii middot 2010) Thus
food safety is cun-entl y an increasing important pub lic health issue laquougovenunents all over
the world are intensify ing the ir efforts to improve food safety
Among the majo r foodbome illness caused by mi croorgan ism are Salmonellosis
Campylobacteriosis Listeriosis and infectio ns due to entero haemonha gic (caJing intes tinal
bleeding) E coli (WHO 2007) III recent years Vibriosis had emerged to be an impOItant
bac te rial disease that canses human intestinal and ext ra-intestinal infections a ll over the world
(Chan elol 2002 Garc ia el al 2009 and Harth et al 2009) The three Vibrio species o f
primary significance in foodbo rne illnesses are V cholerae (serogroups 01 non-Ol and
2
- bull i
0 139) V vulnijiclIs and V paralwemolyricus Presently gast roenteitis caused by V
parahaemolyticus is extensively documented due to its increasing outbreaks (Alam el 01
2003 Gwendelynne el 01 2005 Gorny 2006 Harth et 01 2009 Roque et 01 2009)
V parahaemolyriclls is widely present in brackish and marine waters and infection to
human is regula rl y associated with the consumption of contaminated raw or undercooked
scafood (Guoxia ng el 01 2009) Ea rli er studies also demonstrated that V parahoemolylicus
can be easily iso lated from costal water sediments suspended particles and plankton due to
its halophi lic characteristic (Marlina el 01 2007 Zinuneman el 01 2007 Zu lkifli el al
2009 Vimala et 01 2010) However recent foodbome outbreaks throughout the world have
been intensive ly linked to consu mption of fresh fruits vegetables and unpasteurized juices
(Gorny 2006) Most recently Tunu ng et 01 (20 10) had rep0l1ed th at the vegetable samples
from supermarkets and wet markets in Selangor Malaysia were posit ive for pathogeni c
species of V parahaemolylicls Conversely there is no ot her known documented research on
the prevalence of this species in vegetables
In this st udy vegetables from two wet markets and two supem1arkets in Kuching
Sa rawak were sampled for the presence of V parallOemolyliclis The sa moles underwent
selective enrichment with alkaline peptone water (APW) supp lemented with 3 NaCI Then
presumptive V parahaemolYlicus were iso lated using TeES agar and CH ROMagar VibrioT
Following isolat ion gram stai ning together with a series of biochemica l tests were perfonned
to assist in the identification and confirmation of the iso lates
3
- bull i ~
IZ Objectives
This study was underta ken with the fo llowing objecti ves
I To isolate presumptive V parahaemolYlicus from marketed vegetabl es samples
in Kuching Sarawak by using Te BS and CHROMagar VibrioTM
II To confirm th e suspected V parahaemolyficus iso lates from marketed
vegetables samples in Kuching Sarawak by usi ng Gram stain and biochemical
tests
4
- bull i ~
USI KhJtlmal Makumat U umk VMVl~m ItLAYSlA AAAWAK
CHAPTER 2
LITERATURE REVIEW
21 The genus Vibrio
The genus Vibrio belongs to the fami ly Vibrionaceae and it consists of at least 34 recogn ized
species (HPA 2007a) In general Vibrio spp are Gram-negat ive fac ulta tively anaerobic
mOlile curved rods bacteria with a single polar flagellum (FEHD 2005) In surface waters of
the world Vibrios are among the mos t common surface organisms (Adeleye et aI 20 J 0)
They are present in both marine and fresh wa ter habiwts and commonly associa ted with
aquatic animals Vibrio pp such as V choerae V parahaemoylicus V vu nificus V
aginoylieus V mimicus V luvia lis V filmissii V melselmikovii V tollisae and V
dalltsea are common human pathogens (Tison 1999 Fi nkelstein 2000) T hey account for a
significant proportion of human infections as most of these vibrios secrete enterotoxins to
foods water or in the gastrointestinal tract (Nishibuchi and DePao la 2005)
22 Vibrio paralJlell1oytictls
Vibrio parahaemoy licus which is formerly known as Pasteurella parahaeliloytlca is a
non-sporing Gram-negat ive facu ltatively-anaerobic bacterium (Curti s el 01 2003) This
5
- ~
halophilic bacterium inhabits warm estuarine waters world wide (Bilung el al 2005) Thus il
reqlllres salt to survive and appears in high er concentrations during the warmer summer
months
The optimum temperature fo r the grow th of V parahaemolylicus is 37 degC and at Ihis
optimum temperature the bac terium has generation times of8 to 9 minutes in water and 12 [ 0
18 minutes in seafood (Montville and Matthews 2008) Thi s bacterium is the most IKeiy
Vibrio species to be imp licated in food borne diseases although both V vulnificus and V
cholera may also causes food bom e infections (Lawley e l al 2008) Clinical isolates of V
parrhaemolyticus produce beta type hemolysis on blood aga r (Wagatsuma agar) The
hemolysis is ca ll ed the Kanagawa-phenomenon (KP) which is associated with the production
ofa thermo stabl e d irect hemolys inTOH (Boyd 2008) TOH caused damage to the euka ryotic
cells by acting as a pore fonning toxin that alters the ion balance of cells (Boyd 2008)
Figure 21 SEM observa tlOos of V plrh cm)JJ(cu~
Image source Belas amp Colwe ll ( 1982)
6
-- UI
23 Fresh vegetables
Consumpti on of raw vegetables is common throughout the world not onl y owing to Ihe health
benefit but they are thought to be free from disease causing pathogens However recent food
borne outbreaks worldwi de is associ ated to consumption of fresh frui ts uncooked vegetables
and unpasteurized juices (Gorny 2006) Fruits and vegetable especially those that are eaten
raw without peeling can be the vehicles for transmission of a wide range of mi croorganis m
(Erdogru l and Sener 2005)
Based on Haeton and Jones (2008) E coli Salmonella spp and Listeria spp are
commonly present in vegetable samples Meldrum el 01 (2009) report ed that the two recent
large ou tbreaks in the United Kingdom showed signi fi cant health problems that cou ld ari se
from consumption of contaminated salad Another study of the preva lence of pathogens in
vegetables was reported by Little el 01 (2007) in which they found Listeria monocylOgenes in
ready-to-eat mixed salads in the United Kingdom Several other studi es on d ifferent
microorga nism isolated from vegetabl es were also report ed recent ly by local researchers
(Chai el 01 2008 Han el 01 2009 Ponni ah el 0 2009)
In Mala ys ia the presence of V parahoemo yticus has also been repOIt ed in raw
vegetables samples from wet markets and supeLl11arkets in Selangor (Tunung et 0 20 I 0)
This same study discovered that raw vegetabl es such as OenGnthe st l7i(era (Japanese
parsley) and C[cumis sali vus (Cucumber) were contaminated with virulent V
7
parahaemoiYlicus Consequen tly these vegetables could act as a transmission route and
poses risk to consumers after consumption Tunung el 01 (2011) had also demonstrated that
the vegetable samples fro m wet market contained higher levels of V paraiJaemolylicls as
compared w ith supennarkets The research suggested that there are many factors that can
contri bu te to the distribution of V parahaemoiylicus such as temperature holding ti me as
well as hygiene and sanitation practice (Tunung el aI 2011) However there is no known
documented research on V parahaemolYlicus in raw vegetables in Sarawak
24 Clinical characteristic of V parahaelllolyliclis infections
II parahaemoiyticus causes three distinct illness syndromes which are gastroenteritis wound
or soft tissues infections and septicemia (Sillljee 2007) The most freq uen t syndrome is
gastroeuteritis The symptoms include dianhea with abdomina l cramps nausea vo miting
headache and low-grade fever (S tarlin 2005 WilU1 and Konema n 2006 Montville and
Matthew 2008) Sometimes the diarrhea is bloody w ith stools described as meat washed
since tl1e stool is reddish and watery (Qadri el aI 2009)
As for wound or tis sue in fections this coudition occurs by the exposure of broken skin
or wound to warm seawater and seafood dripp ings (Ralph and Currie 2007 Simjee 2007)
In severe cases serious wound infection will resu lt in necrotizing fasc iiti s (Ralph and Curri e
2007) Although less common in V paraiwemolyliclIs infection septicemia is characteri7cd
by fever or hypotension with the isolation of V parahaemolyticus in the blood Septicemia
typicall y occurs in person with underlying health problems such as liver disease diabetes
cancer or other immune disorder (Simjee 2007) V parahaemoyticlis can even ca use fala l
septicemia in an immune-compromised hosts
The symptoms usuall y occur on the average of 15 hours but can began as early as 4
hours and as late as 36 hours after exposure and this will continue up to 3 days (Barceloux
200S) However these illnesses are usua lly categorized as a short illnesses and no prolonged
enect will take place after recovery (Wright et al 2009)
25 Isolation of V parahael1lolyliclIs
For the isolation of V parahaemoyticus an initial emichment using alka line peptone wat er
(APW) with pH S5 would be useful as this medium supports the growth uf viable ce lls
(Simjee 2007) Plati ng of emichment broth onto TCBS agar has been widely used for
selective isolation of V parahaemolytics from foods (Thompson el ai 2006 Oi Pinto et al
201 0 Canizalez -Roman et ai 2011 ) An average of 24 hours incubation at 37 degC is requ ired
fo r typica l V parahaemolylicus to develop colonies that are smooth gree n (s u~rose nega tive)
and 2 to 3 nun in di ameter on TCBS (ISO 2007) However V parahaenlOlytlcus co lonies on
TCBS agar are difficult to distinguish vi suall y from other bacterial colonies since they can be
covered by yellow color produced by sucrose-fennenting bacteria (Thompson el 01 2006
Rambach 2007)
9
_ ------~-- i ~~
Another widely used selective medium is CHROMagar VibrioT On this medium V
pllrahaemoiy liclis will developed mau ve color colonies (Kudo et aI 2001 Oi Pi nto 2010
Canizalez-Roman 2011) This agar medium contains substrates for beta-galactosidase which
was developed specifically to differentiate V parahaemolytics from ot her bacteria by us ing a
chromogenic su bstrate instead of sugar fermentation in traditional growth med ia such as
TCBS (Hara-Kudo et 01 2001) Therefore it is more sensitiv e and accurate for identi fying V
porahllemolyticlIs in food samples rather than the use of Te ES (Oi Pinto 2010 Canizalez-
Roman 20 II)
26 Identification through Gram-stain and biochemical tests
V parahaemoiyticus is a Gram-negative bacterium th us this organi sm stained pink or red
after Gram staining (HPA 20073) Under microscopic observation a typical V
pllrahaemolyticus appeared as rod characterist ica lly curved or comma-shoped The bacterium
is also positi ve for motility under microscopic observations (ISO 2007 BPA 2007a)
V parahaemolylieus ca n be identified through a seri es of biochemical tests According
to Khan et 01 (2007) V para haemolyticus can be distinct ively differentia ted fro m the other
Vibrio spp by performing sa lt tolerance and acid fro m femlen tation tests A typical V
parahaemoiYlicus co lonies would not grow in AFW without th e presence of at least 3 of sali
10
~-------
but it can only endure the presence of salt up to 8 (ISO 2007 Khan el aI 2007) In the
other hand the fermentation test for acid production is the most important test to differentiate
between V parahaemolyliclIs and V vulnificus species (Khan el aI 2007) Jayasinge et ai
(2010) even suggested that the combination of salt toleranc e test Voges-Proskauer test and
ONPG test can provide a strai ghtforward differentiation of V parahaemoly liclis from the
other Vibrionaceae species (Figure 31)
Citrate test screens the ab ilit y of bacterial isolates to utilize ci trate as its carbon and
energy source (MacFaddin 2000) This test is a very important test to identify gram-negative
pathogens (Tang el aI 1998) The MR-VP medium which was used for Met hyl red and
Voges Proskauer test contains glucose and peptone Methyl red test screens for the bacterial
metaboli sm pathways for glucose fermentation while the Voges-Proskauer tes t is an assay for
organisms which ferment glucose with only one fermentation product usuall y acetic acid
The TS[ test is another useful test tha t can distinguish V parahaemolyticus from other
bacteria (ISO 2007) The TSJ agar med ium contains peptone fenous sulfate lactose sucrose
and a small amount of glucose (dex trose) The pH indi cator is phenol red This medium
differentiates bacteria based on their ability to fennent glucose lactose ando r sucrose and on
their abi lity to reduce sulfur to hydrogen sulfide (H S) The T SJ test also haj the abi lity to
determine the production of H]S (Kim et aI 1999)
In additi on other bi ochemica l tests such as oxidase lysine decarboxylase om ithi ne
decarboxylase ca talase and indole can also be used for the purpose of identifying V
II
-- ---- - ----------- - ~i
parahaemoyticus iso lates (ISO 2007 Jayasinghe et ai 2007 Khan et aI 2007) The
biochemical reactions o f V parahaemoiyticus are summarized in Table 21
Table 21 Siandard biochemical tesl reactions of V porahaemoyticus
TeSi S Reaction of V parahaem olyaclIs Refe rences Salt tol ance
+
Sali ne TSI agar KIA
No HS and gas production
Simmons Citrnh
Voges-Proskauer
Methyl-Red +
[SO (2007)
Jayasinge et al (2007)
Khan et of (2007)
ISO (2007)
Kim et I (1999)
Abide et til (1999)
Kim 11111 (1999)
Khan et til (2007)
Jayasinge el III (20 (0)
Kim et 0 (2 007)
Note (-) ~ nega live (+) ~ positi ve K ~ alka line ~ aC idic
12
CHAPT ER 3
MATERIALS AND METI IODS
31 Samples collection
Eighty raw vegetable samples were collected week ly from two wet markets (WI amp W2) and
two supennarkets (S 1 amp S2) in Kuching Sarawa k The collected samples consisted o f four
di fferent groups of vegetables which are regularly consumed ra w by the locals These samples
wcr~ rand omly selected within ten weeks -period which is from October 20 10 to January
20 11 Duri ng collection each sample was transferred to clean plastic bags labeled acco rding
to the place they were collected and were analyzed immed iately on arriva l to the labo rat ory
The types of raw vegetable sa mples obtained were as shown in Table 31
Tab le 31 Types o f raw vegetable samples co llected from wet markes and supcmlarkels
Group Loca l nam e English nam e Scienti fi c Home l mple coding No or samples
Soil Kubis Cabbage Brassica v eracea K 20 Roots Lobak Canmiddotot Douclfs carola C ~O
mera h Beans KaCtlO g Four-winged PsophOCaJjJ lls KB cO
bow l bean JeIlagulIolobus Shoot Tauge Bean-I_prout Vigna rad mc T 10 Total 80
13
---------~ I I
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
Tbe Incidence of Vibrio p arahaellloly ticlis in Raw Vegetables at Local Wet Markets and Supermarkets in Kuching Sarawak
Sabatini lihob
Resource BIOIClhnoltlg) Progrtlm me Facu Ity or Resource Science Bud T~lln(llogy
Uni versity Malaysb Sa rawa k
ABSTRACT
Vibrio parawemoyliCLls is a foodbome pa thogen that is regular ly assoc iated with the consu mption o f
raw or undercooked seafood The mos t frequent sympto m of V parahaemoylicus infecr ion is
gastroenteritis Recen tly this pa thogen was also disco vered in raw vegetable samples in Peninsular
Malaysia Thus the aim of this study is to isolate and de tect V paahaemoylicu~ in raw vegdables T he samples which consisted of carrot fourmiddot winged bean cabbage and bea n sp ro ul were purc hased
tiom two wet markets and two supenn arkets in Kucll ing Sarawak In th is stud y alkaline peplone
water (A PW) wa s used as the enridunent medium The combinati o n o f two types o f selective agar
TCBS and CHROMa gar Vibrio nbull1 was successfully app lied to isolate the bacteria A fte r a series of
biochemical tests only 2 out of the 80 samples were pos itive for the p resence of V parahaemoylicLls
Although the peroentage of V pOrehaemolylics prevalence was low (2 5) the resu lts h ighli ghted
the fac t that raw vege tables could also be contaminated w It h V parahaenro~Vlicus
Key words Vib rio parahemotyfic~ raw vegetables TC BS and CHROMagar Vibrio rM
ABSTRAK
Vibrio parawcmoylicus merupakan sejenis patogen bmvaan makanafl )long sering dikairkan dengan
pengambiall makonon laul seewa men fa h Tonda paling kemp bagi j angkitall k parahwnJ r lvticlfs
ialah gasroenlritis TerbolU pafogen ini Ina dijumpa i di dalam sUlllp~1 scJyuran melJfah di
Semenanjung A1nlaYiia Kajial1 ini berlujuan un uk mengasmg dan mengesan V nanlhaemolliqi di
daall1 sampel $oyuran menfah Sampel yang lerdiri doripada obok merah kac(mg botol kobis dan
(l uge teah dlbeli dari dU(J pasai basah Clnd clua pasar raya di Kuchi lg Sorm lak Il l pepton beralktrli
(A PW) fel(lh digul1akan Ill1tuk memperbonyakko1 jwn Jah bakfcna Kombilw si pelggunaon agar
terpilih Tess dan CHROkiagar Vibrio T1 tdoh bejaya mel11ellcilkon ba( ur ia yallg dikehendoki
Selelah beberapa siri lljion biokimia didapafi 2 daripada 80 sampel relah dlcemQj oleh 1 rar(fhoenlJliJji(u~ Mcsklpun perafus kehadiran V norahaemolvticus adalah remJah (25~ o)
kepuflfsan kajian ini menunjukkan bahawo saYllran mental boleh dicemari ole Y paraha~moh IIcm
Kata klllt ci Vibrio oorqhaemol liclfs sayuran men fah TCBS dOll CHROtviagar Vihriotrade
-
CHAPTER 1
INTRODUCTION
11 Introduction
Foodbome illness assoc iated with consumption of contam inated food is a corrunon hea lth
problem that occurs worldwide The global inc idence of food bo rne ill ness is d ifficu lt to
estimate but it has been reported that in 2005 alone around 76 million cases of food bo rne
diseases resu lting in 325000 hospita liza tions and 5000 dea ths are es timated to occur in the
United State (WHO 20 11 ) In addition food contaminat ion also causes enormous socia l and
economical burden on the community A consumer research repon released on 2010 revea led
that the healt h-related costs of food-borne illnesses had cost up to 152 billion do llar a year
includ ing the costs of med ica l bills lost wages and lost produc tivi ty (Smii middot 2010) Thus
food safety is cun-entl y an increasing important pub lic health issue laquougovenunents all over
the world are intensify ing the ir efforts to improve food safety
Among the majo r foodbome illness caused by mi croorgan ism are Salmonellosis
Campylobacteriosis Listeriosis and infectio ns due to entero haemonha gic (caJing intes tinal
bleeding) E coli (WHO 2007) III recent years Vibriosis had emerged to be an impOItant
bac te rial disease that canses human intestinal and ext ra-intestinal infections a ll over the world
(Chan elol 2002 Garc ia el al 2009 and Harth et al 2009) The three Vibrio species o f
primary significance in foodbo rne illnesses are V cholerae (serogroups 01 non-Ol and
2
- bull i
0 139) V vulnijiclIs and V paralwemolyricus Presently gast roenteitis caused by V
parahaemolyticus is extensively documented due to its increasing outbreaks (Alam el 01
2003 Gwendelynne el 01 2005 Gorny 2006 Harth et 01 2009 Roque et 01 2009)
V parahaemolyriclls is widely present in brackish and marine waters and infection to
human is regula rl y associated with the consumption of contaminated raw or undercooked
scafood (Guoxia ng el 01 2009) Ea rli er studies also demonstrated that V parahoemolylicus
can be easily iso lated from costal water sediments suspended particles and plankton due to
its halophi lic characteristic (Marlina el 01 2007 Zinuneman el 01 2007 Zu lkifli el al
2009 Vimala et 01 2010) However recent foodbome outbreaks throughout the world have
been intensive ly linked to consu mption of fresh fruits vegetables and unpasteurized juices
(Gorny 2006) Most recently Tunu ng et 01 (20 10) had rep0l1ed th at the vegetable samples
from supermarkets and wet markets in Selangor Malaysia were posit ive for pathogeni c
species of V parahaemolylicls Conversely there is no ot her known documented research on
the prevalence of this species in vegetables
In this st udy vegetables from two wet markets and two supem1arkets in Kuching
Sa rawak were sampled for the presence of V parallOemolyliclis The sa moles underwent
selective enrichment with alkaline peptone water (APW) supp lemented with 3 NaCI Then
presumptive V parahaemolYlicus were iso lated using TeES agar and CH ROMagar VibrioT
Following isolat ion gram stai ning together with a series of biochemica l tests were perfonned
to assist in the identification and confirmation of the iso lates
3
- bull i ~
IZ Objectives
This study was underta ken with the fo llowing objecti ves
I To isolate presumptive V parahaemolYlicus from marketed vegetabl es samples
in Kuching Sarawak by using Te BS and CHROMagar VibrioTM
II To confirm th e suspected V parahaemolyficus iso lates from marketed
vegetables samples in Kuching Sarawak by usi ng Gram stain and biochemical
tests
4
- bull i ~
USI KhJtlmal Makumat U umk VMVl~m ItLAYSlA AAAWAK
CHAPTER 2
LITERATURE REVIEW
21 The genus Vibrio
The genus Vibrio belongs to the fami ly Vibrionaceae and it consists of at least 34 recogn ized
species (HPA 2007a) In general Vibrio spp are Gram-negat ive fac ulta tively anaerobic
mOlile curved rods bacteria with a single polar flagellum (FEHD 2005) In surface waters of
the world Vibrios are among the mos t common surface organisms (Adeleye et aI 20 J 0)
They are present in both marine and fresh wa ter habiwts and commonly associa ted with
aquatic animals Vibrio pp such as V choerae V parahaemoylicus V vu nificus V
aginoylieus V mimicus V luvia lis V filmissii V melselmikovii V tollisae and V
dalltsea are common human pathogens (Tison 1999 Fi nkelstein 2000) T hey account for a
significant proportion of human infections as most of these vibrios secrete enterotoxins to
foods water or in the gastrointestinal tract (Nishibuchi and DePao la 2005)
22 Vibrio paralJlell1oytictls
Vibrio parahaemoy licus which is formerly known as Pasteurella parahaeliloytlca is a
non-sporing Gram-negat ive facu ltatively-anaerobic bacterium (Curti s el 01 2003) This
5
- ~
halophilic bacterium inhabits warm estuarine waters world wide (Bilung el al 2005) Thus il
reqlllres salt to survive and appears in high er concentrations during the warmer summer
months
The optimum temperature fo r the grow th of V parahaemolylicus is 37 degC and at Ihis
optimum temperature the bac terium has generation times of8 to 9 minutes in water and 12 [ 0
18 minutes in seafood (Montville and Matthews 2008) Thi s bacterium is the most IKeiy
Vibrio species to be imp licated in food borne diseases although both V vulnificus and V
cholera may also causes food bom e infections (Lawley e l al 2008) Clinical isolates of V
parrhaemolyticus produce beta type hemolysis on blood aga r (Wagatsuma agar) The
hemolysis is ca ll ed the Kanagawa-phenomenon (KP) which is associated with the production
ofa thermo stabl e d irect hemolys inTOH (Boyd 2008) TOH caused damage to the euka ryotic
cells by acting as a pore fonning toxin that alters the ion balance of cells (Boyd 2008)
Figure 21 SEM observa tlOos of V plrh cm)JJ(cu~
Image source Belas amp Colwe ll ( 1982)
6
-- UI
23 Fresh vegetables
Consumpti on of raw vegetables is common throughout the world not onl y owing to Ihe health
benefit but they are thought to be free from disease causing pathogens However recent food
borne outbreaks worldwi de is associ ated to consumption of fresh frui ts uncooked vegetables
and unpasteurized juices (Gorny 2006) Fruits and vegetable especially those that are eaten
raw without peeling can be the vehicles for transmission of a wide range of mi croorganis m
(Erdogru l and Sener 2005)
Based on Haeton and Jones (2008) E coli Salmonella spp and Listeria spp are
commonly present in vegetable samples Meldrum el 01 (2009) report ed that the two recent
large ou tbreaks in the United Kingdom showed signi fi cant health problems that cou ld ari se
from consumption of contaminated salad Another study of the preva lence of pathogens in
vegetables was reported by Little el 01 (2007) in which they found Listeria monocylOgenes in
ready-to-eat mixed salads in the United Kingdom Several other studi es on d ifferent
microorga nism isolated from vegetabl es were also report ed recent ly by local researchers
(Chai el 01 2008 Han el 01 2009 Ponni ah el 0 2009)
In Mala ys ia the presence of V parahoemo yticus has also been repOIt ed in raw
vegetables samples from wet markets and supeLl11arkets in Selangor (Tunung et 0 20 I 0)
This same study discovered that raw vegetabl es such as OenGnthe st l7i(era (Japanese
parsley) and C[cumis sali vus (Cucumber) were contaminated with virulent V
7
parahaemoiYlicus Consequen tly these vegetables could act as a transmission route and
poses risk to consumers after consumption Tunung el 01 (2011) had also demonstrated that
the vegetable samples fro m wet market contained higher levels of V paraiJaemolylicls as
compared w ith supennarkets The research suggested that there are many factors that can
contri bu te to the distribution of V parahaemoiylicus such as temperature holding ti me as
well as hygiene and sanitation practice (Tunung el aI 2011) However there is no known
documented research on V parahaemolYlicus in raw vegetables in Sarawak
24 Clinical characteristic of V parahaelllolyliclis infections
II parahaemoiyticus causes three distinct illness syndromes which are gastroenteritis wound
or soft tissues infections and septicemia (Sillljee 2007) The most freq uen t syndrome is
gastroeuteritis The symptoms include dianhea with abdomina l cramps nausea vo miting
headache and low-grade fever (S tarlin 2005 WilU1 and Konema n 2006 Montville and
Matthew 2008) Sometimes the diarrhea is bloody w ith stools described as meat washed
since tl1e stool is reddish and watery (Qadri el aI 2009)
As for wound or tis sue in fections this coudition occurs by the exposure of broken skin
or wound to warm seawater and seafood dripp ings (Ralph and Currie 2007 Simjee 2007)
In severe cases serious wound infection will resu lt in necrotizing fasc iiti s (Ralph and Curri e
2007) Although less common in V paraiwemolyliclIs infection septicemia is characteri7cd
by fever or hypotension with the isolation of V parahaemolyticus in the blood Septicemia
typicall y occurs in person with underlying health problems such as liver disease diabetes
cancer or other immune disorder (Simjee 2007) V parahaemoyticlis can even ca use fala l
septicemia in an immune-compromised hosts
The symptoms usuall y occur on the average of 15 hours but can began as early as 4
hours and as late as 36 hours after exposure and this will continue up to 3 days (Barceloux
200S) However these illnesses are usua lly categorized as a short illnesses and no prolonged
enect will take place after recovery (Wright et al 2009)
25 Isolation of V parahael1lolyliclIs
For the isolation of V parahaemoyticus an initial emichment using alka line peptone wat er
(APW) with pH S5 would be useful as this medium supports the growth uf viable ce lls
(Simjee 2007) Plati ng of emichment broth onto TCBS agar has been widely used for
selective isolation of V parahaemolytics from foods (Thompson el ai 2006 Oi Pinto et al
201 0 Canizalez -Roman et ai 2011 ) An average of 24 hours incubation at 37 degC is requ ired
fo r typica l V parahaemolylicus to develop colonies that are smooth gree n (s u~rose nega tive)
and 2 to 3 nun in di ameter on TCBS (ISO 2007) However V parahaenlOlytlcus co lonies on
TCBS agar are difficult to distinguish vi suall y from other bacterial colonies since they can be
covered by yellow color produced by sucrose-fennenting bacteria (Thompson el 01 2006
Rambach 2007)
9
_ ------~-- i ~~
Another widely used selective medium is CHROMagar VibrioT On this medium V
pllrahaemoiy liclis will developed mau ve color colonies (Kudo et aI 2001 Oi Pi nto 2010
Canizalez-Roman 2011) This agar medium contains substrates for beta-galactosidase which
was developed specifically to differentiate V parahaemolytics from ot her bacteria by us ing a
chromogenic su bstrate instead of sugar fermentation in traditional growth med ia such as
TCBS (Hara-Kudo et 01 2001) Therefore it is more sensitiv e and accurate for identi fying V
porahllemolyticlIs in food samples rather than the use of Te ES (Oi Pinto 2010 Canizalez-
Roman 20 II)
26 Identification through Gram-stain and biochemical tests
V parahaemoiyticus is a Gram-negative bacterium th us this organi sm stained pink or red
after Gram staining (HPA 20073) Under microscopic observation a typical V
pllrahaemolyticus appeared as rod characterist ica lly curved or comma-shoped The bacterium
is also positi ve for motility under microscopic observations (ISO 2007 BPA 2007a)
V parahaemolylieus ca n be identified through a seri es of biochemical tests According
to Khan et 01 (2007) V para haemolyticus can be distinct ively differentia ted fro m the other
Vibrio spp by performing sa lt tolerance and acid fro m femlen tation tests A typical V
parahaemoiYlicus co lonies would not grow in AFW without th e presence of at least 3 of sali
10
~-------
but it can only endure the presence of salt up to 8 (ISO 2007 Khan el aI 2007) In the
other hand the fermentation test for acid production is the most important test to differentiate
between V parahaemolyliclIs and V vulnificus species (Khan el aI 2007) Jayasinge et ai
(2010) even suggested that the combination of salt toleranc e test Voges-Proskauer test and
ONPG test can provide a strai ghtforward differentiation of V parahaemoly liclis from the
other Vibrionaceae species (Figure 31)
Citrate test screens the ab ilit y of bacterial isolates to utilize ci trate as its carbon and
energy source (MacFaddin 2000) This test is a very important test to identify gram-negative
pathogens (Tang el aI 1998) The MR-VP medium which was used for Met hyl red and
Voges Proskauer test contains glucose and peptone Methyl red test screens for the bacterial
metaboli sm pathways for glucose fermentation while the Voges-Proskauer tes t is an assay for
organisms which ferment glucose with only one fermentation product usuall y acetic acid
The TS[ test is another useful test tha t can distinguish V parahaemolyticus from other
bacteria (ISO 2007) The TSJ agar med ium contains peptone fenous sulfate lactose sucrose
and a small amount of glucose (dex trose) The pH indi cator is phenol red This medium
differentiates bacteria based on their ability to fennent glucose lactose ando r sucrose and on
their abi lity to reduce sulfur to hydrogen sulfide (H S) The T SJ test also haj the abi lity to
determine the production of H]S (Kim et aI 1999)
In additi on other bi ochemica l tests such as oxidase lysine decarboxylase om ithi ne
decarboxylase ca talase and indole can also be used for the purpose of identifying V
II
-- ---- - ----------- - ~i
parahaemoyticus iso lates (ISO 2007 Jayasinghe et ai 2007 Khan et aI 2007) The
biochemical reactions o f V parahaemoiyticus are summarized in Table 21
Table 21 Siandard biochemical tesl reactions of V porahaemoyticus
TeSi S Reaction of V parahaem olyaclIs Refe rences Salt tol ance
+
Sali ne TSI agar KIA
No HS and gas production
Simmons Citrnh
Voges-Proskauer
Methyl-Red +
[SO (2007)
Jayasinge et al (2007)
Khan et of (2007)
ISO (2007)
Kim et I (1999)
Abide et til (1999)
Kim 11111 (1999)
Khan et til (2007)
Jayasinge el III (20 (0)
Kim et 0 (2 007)
Note (-) ~ nega live (+) ~ positi ve K ~ alka line ~ aC idic
12
CHAPT ER 3
MATERIALS AND METI IODS
31 Samples collection
Eighty raw vegetable samples were collected week ly from two wet markets (WI amp W2) and
two supennarkets (S 1 amp S2) in Kuching Sarawa k The collected samples consisted o f four
di fferent groups of vegetables which are regularly consumed ra w by the locals These samples
wcr~ rand omly selected within ten weeks -period which is from October 20 10 to January
20 11 Duri ng collection each sample was transferred to clean plastic bags labeled acco rding
to the place they were collected and were analyzed immed iately on arriva l to the labo rat ory
The types of raw vegetable sa mples obtained were as shown in Table 31
Tab le 31 Types o f raw vegetable samples co llected from wet markes and supcmlarkels
Group Loca l nam e English nam e Scienti fi c Home l mple coding No or samples
Soil Kubis Cabbage Brassica v eracea K 20 Roots Lobak Canmiddotot Douclfs carola C ~O
mera h Beans KaCtlO g Four-winged PsophOCaJjJ lls KB cO
bow l bean JeIlagulIolobus Shoot Tauge Bean-I_prout Vigna rad mc T 10 Total 80
13
---------~ I I
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
CHAPTER 1
INTRODUCTION
11 Introduction
Foodbome illness assoc iated with consumption of contam inated food is a corrunon hea lth
problem that occurs worldwide The global inc idence of food bo rne ill ness is d ifficu lt to
estimate but it has been reported that in 2005 alone around 76 million cases of food bo rne
diseases resu lting in 325000 hospita liza tions and 5000 dea ths are es timated to occur in the
United State (WHO 20 11 ) In addition food contaminat ion also causes enormous socia l and
economical burden on the community A consumer research repon released on 2010 revea led
that the healt h-related costs of food-borne illnesses had cost up to 152 billion do llar a year
includ ing the costs of med ica l bills lost wages and lost produc tivi ty (Smii middot 2010) Thus
food safety is cun-entl y an increasing important pub lic health issue laquougovenunents all over
the world are intensify ing the ir efforts to improve food safety
Among the majo r foodbome illness caused by mi croorgan ism are Salmonellosis
Campylobacteriosis Listeriosis and infectio ns due to entero haemonha gic (caJing intes tinal
bleeding) E coli (WHO 2007) III recent years Vibriosis had emerged to be an impOItant
bac te rial disease that canses human intestinal and ext ra-intestinal infections a ll over the world
(Chan elol 2002 Garc ia el al 2009 and Harth et al 2009) The three Vibrio species o f
primary significance in foodbo rne illnesses are V cholerae (serogroups 01 non-Ol and
2
- bull i
0 139) V vulnijiclIs and V paralwemolyricus Presently gast roenteitis caused by V
parahaemolyticus is extensively documented due to its increasing outbreaks (Alam el 01
2003 Gwendelynne el 01 2005 Gorny 2006 Harth et 01 2009 Roque et 01 2009)
V parahaemolyriclls is widely present in brackish and marine waters and infection to
human is regula rl y associated with the consumption of contaminated raw or undercooked
scafood (Guoxia ng el 01 2009) Ea rli er studies also demonstrated that V parahoemolylicus
can be easily iso lated from costal water sediments suspended particles and plankton due to
its halophi lic characteristic (Marlina el 01 2007 Zinuneman el 01 2007 Zu lkifli el al
2009 Vimala et 01 2010) However recent foodbome outbreaks throughout the world have
been intensive ly linked to consu mption of fresh fruits vegetables and unpasteurized juices
(Gorny 2006) Most recently Tunu ng et 01 (20 10) had rep0l1ed th at the vegetable samples
from supermarkets and wet markets in Selangor Malaysia were posit ive for pathogeni c
species of V parahaemolylicls Conversely there is no ot her known documented research on
the prevalence of this species in vegetables
In this st udy vegetables from two wet markets and two supem1arkets in Kuching
Sa rawak were sampled for the presence of V parallOemolyliclis The sa moles underwent
selective enrichment with alkaline peptone water (APW) supp lemented with 3 NaCI Then
presumptive V parahaemolYlicus were iso lated using TeES agar and CH ROMagar VibrioT
Following isolat ion gram stai ning together with a series of biochemica l tests were perfonned
to assist in the identification and confirmation of the iso lates
3
- bull i ~
IZ Objectives
This study was underta ken with the fo llowing objecti ves
I To isolate presumptive V parahaemolYlicus from marketed vegetabl es samples
in Kuching Sarawak by using Te BS and CHROMagar VibrioTM
II To confirm th e suspected V parahaemolyficus iso lates from marketed
vegetables samples in Kuching Sarawak by usi ng Gram stain and biochemical
tests
4
- bull i ~
USI KhJtlmal Makumat U umk VMVl~m ItLAYSlA AAAWAK
CHAPTER 2
LITERATURE REVIEW
21 The genus Vibrio
The genus Vibrio belongs to the fami ly Vibrionaceae and it consists of at least 34 recogn ized
species (HPA 2007a) In general Vibrio spp are Gram-negat ive fac ulta tively anaerobic
mOlile curved rods bacteria with a single polar flagellum (FEHD 2005) In surface waters of
the world Vibrios are among the mos t common surface organisms (Adeleye et aI 20 J 0)
They are present in both marine and fresh wa ter habiwts and commonly associa ted with
aquatic animals Vibrio pp such as V choerae V parahaemoylicus V vu nificus V
aginoylieus V mimicus V luvia lis V filmissii V melselmikovii V tollisae and V
dalltsea are common human pathogens (Tison 1999 Fi nkelstein 2000) T hey account for a
significant proportion of human infections as most of these vibrios secrete enterotoxins to
foods water or in the gastrointestinal tract (Nishibuchi and DePao la 2005)
22 Vibrio paralJlell1oytictls
Vibrio parahaemoy licus which is formerly known as Pasteurella parahaeliloytlca is a
non-sporing Gram-negat ive facu ltatively-anaerobic bacterium (Curti s el 01 2003) This
5
- ~
halophilic bacterium inhabits warm estuarine waters world wide (Bilung el al 2005) Thus il
reqlllres salt to survive and appears in high er concentrations during the warmer summer
months
The optimum temperature fo r the grow th of V parahaemolylicus is 37 degC and at Ihis
optimum temperature the bac terium has generation times of8 to 9 minutes in water and 12 [ 0
18 minutes in seafood (Montville and Matthews 2008) Thi s bacterium is the most IKeiy
Vibrio species to be imp licated in food borne diseases although both V vulnificus and V
cholera may also causes food bom e infections (Lawley e l al 2008) Clinical isolates of V
parrhaemolyticus produce beta type hemolysis on blood aga r (Wagatsuma agar) The
hemolysis is ca ll ed the Kanagawa-phenomenon (KP) which is associated with the production
ofa thermo stabl e d irect hemolys inTOH (Boyd 2008) TOH caused damage to the euka ryotic
cells by acting as a pore fonning toxin that alters the ion balance of cells (Boyd 2008)
Figure 21 SEM observa tlOos of V plrh cm)JJ(cu~
Image source Belas amp Colwe ll ( 1982)
6
-- UI
23 Fresh vegetables
Consumpti on of raw vegetables is common throughout the world not onl y owing to Ihe health
benefit but they are thought to be free from disease causing pathogens However recent food
borne outbreaks worldwi de is associ ated to consumption of fresh frui ts uncooked vegetables
and unpasteurized juices (Gorny 2006) Fruits and vegetable especially those that are eaten
raw without peeling can be the vehicles for transmission of a wide range of mi croorganis m
(Erdogru l and Sener 2005)
Based on Haeton and Jones (2008) E coli Salmonella spp and Listeria spp are
commonly present in vegetable samples Meldrum el 01 (2009) report ed that the two recent
large ou tbreaks in the United Kingdom showed signi fi cant health problems that cou ld ari se
from consumption of contaminated salad Another study of the preva lence of pathogens in
vegetables was reported by Little el 01 (2007) in which they found Listeria monocylOgenes in
ready-to-eat mixed salads in the United Kingdom Several other studi es on d ifferent
microorga nism isolated from vegetabl es were also report ed recent ly by local researchers
(Chai el 01 2008 Han el 01 2009 Ponni ah el 0 2009)
In Mala ys ia the presence of V parahoemo yticus has also been repOIt ed in raw
vegetables samples from wet markets and supeLl11arkets in Selangor (Tunung et 0 20 I 0)
This same study discovered that raw vegetabl es such as OenGnthe st l7i(era (Japanese
parsley) and C[cumis sali vus (Cucumber) were contaminated with virulent V
7
parahaemoiYlicus Consequen tly these vegetables could act as a transmission route and
poses risk to consumers after consumption Tunung el 01 (2011) had also demonstrated that
the vegetable samples fro m wet market contained higher levels of V paraiJaemolylicls as
compared w ith supennarkets The research suggested that there are many factors that can
contri bu te to the distribution of V parahaemoiylicus such as temperature holding ti me as
well as hygiene and sanitation practice (Tunung el aI 2011) However there is no known
documented research on V parahaemolYlicus in raw vegetables in Sarawak
24 Clinical characteristic of V parahaelllolyliclis infections
II parahaemoiyticus causes three distinct illness syndromes which are gastroenteritis wound
or soft tissues infections and septicemia (Sillljee 2007) The most freq uen t syndrome is
gastroeuteritis The symptoms include dianhea with abdomina l cramps nausea vo miting
headache and low-grade fever (S tarlin 2005 WilU1 and Konema n 2006 Montville and
Matthew 2008) Sometimes the diarrhea is bloody w ith stools described as meat washed
since tl1e stool is reddish and watery (Qadri el aI 2009)
As for wound or tis sue in fections this coudition occurs by the exposure of broken skin
or wound to warm seawater and seafood dripp ings (Ralph and Currie 2007 Simjee 2007)
In severe cases serious wound infection will resu lt in necrotizing fasc iiti s (Ralph and Curri e
2007) Although less common in V paraiwemolyliclIs infection septicemia is characteri7cd
by fever or hypotension with the isolation of V parahaemolyticus in the blood Septicemia
typicall y occurs in person with underlying health problems such as liver disease diabetes
cancer or other immune disorder (Simjee 2007) V parahaemoyticlis can even ca use fala l
septicemia in an immune-compromised hosts
The symptoms usuall y occur on the average of 15 hours but can began as early as 4
hours and as late as 36 hours after exposure and this will continue up to 3 days (Barceloux
200S) However these illnesses are usua lly categorized as a short illnesses and no prolonged
enect will take place after recovery (Wright et al 2009)
25 Isolation of V parahael1lolyliclIs
For the isolation of V parahaemoyticus an initial emichment using alka line peptone wat er
(APW) with pH S5 would be useful as this medium supports the growth uf viable ce lls
(Simjee 2007) Plati ng of emichment broth onto TCBS agar has been widely used for
selective isolation of V parahaemolytics from foods (Thompson el ai 2006 Oi Pinto et al
201 0 Canizalez -Roman et ai 2011 ) An average of 24 hours incubation at 37 degC is requ ired
fo r typica l V parahaemolylicus to develop colonies that are smooth gree n (s u~rose nega tive)
and 2 to 3 nun in di ameter on TCBS (ISO 2007) However V parahaenlOlytlcus co lonies on
TCBS agar are difficult to distinguish vi suall y from other bacterial colonies since they can be
covered by yellow color produced by sucrose-fennenting bacteria (Thompson el 01 2006
Rambach 2007)
9
_ ------~-- i ~~
Another widely used selective medium is CHROMagar VibrioT On this medium V
pllrahaemoiy liclis will developed mau ve color colonies (Kudo et aI 2001 Oi Pi nto 2010
Canizalez-Roman 2011) This agar medium contains substrates for beta-galactosidase which
was developed specifically to differentiate V parahaemolytics from ot her bacteria by us ing a
chromogenic su bstrate instead of sugar fermentation in traditional growth med ia such as
TCBS (Hara-Kudo et 01 2001) Therefore it is more sensitiv e and accurate for identi fying V
porahllemolyticlIs in food samples rather than the use of Te ES (Oi Pinto 2010 Canizalez-
Roman 20 II)
26 Identification through Gram-stain and biochemical tests
V parahaemoiyticus is a Gram-negative bacterium th us this organi sm stained pink or red
after Gram staining (HPA 20073) Under microscopic observation a typical V
pllrahaemolyticus appeared as rod characterist ica lly curved or comma-shoped The bacterium
is also positi ve for motility under microscopic observations (ISO 2007 BPA 2007a)
V parahaemolylieus ca n be identified through a seri es of biochemical tests According
to Khan et 01 (2007) V para haemolyticus can be distinct ively differentia ted fro m the other
Vibrio spp by performing sa lt tolerance and acid fro m femlen tation tests A typical V
parahaemoiYlicus co lonies would not grow in AFW without th e presence of at least 3 of sali
10
~-------
but it can only endure the presence of salt up to 8 (ISO 2007 Khan el aI 2007) In the
other hand the fermentation test for acid production is the most important test to differentiate
between V parahaemolyliclIs and V vulnificus species (Khan el aI 2007) Jayasinge et ai
(2010) even suggested that the combination of salt toleranc e test Voges-Proskauer test and
ONPG test can provide a strai ghtforward differentiation of V parahaemoly liclis from the
other Vibrionaceae species (Figure 31)
Citrate test screens the ab ilit y of bacterial isolates to utilize ci trate as its carbon and
energy source (MacFaddin 2000) This test is a very important test to identify gram-negative
pathogens (Tang el aI 1998) The MR-VP medium which was used for Met hyl red and
Voges Proskauer test contains glucose and peptone Methyl red test screens for the bacterial
metaboli sm pathways for glucose fermentation while the Voges-Proskauer tes t is an assay for
organisms which ferment glucose with only one fermentation product usuall y acetic acid
The TS[ test is another useful test tha t can distinguish V parahaemolyticus from other
bacteria (ISO 2007) The TSJ agar med ium contains peptone fenous sulfate lactose sucrose
and a small amount of glucose (dex trose) The pH indi cator is phenol red This medium
differentiates bacteria based on their ability to fennent glucose lactose ando r sucrose and on
their abi lity to reduce sulfur to hydrogen sulfide (H S) The T SJ test also haj the abi lity to
determine the production of H]S (Kim et aI 1999)
In additi on other bi ochemica l tests such as oxidase lysine decarboxylase om ithi ne
decarboxylase ca talase and indole can also be used for the purpose of identifying V
II
-- ---- - ----------- - ~i
parahaemoyticus iso lates (ISO 2007 Jayasinghe et ai 2007 Khan et aI 2007) The
biochemical reactions o f V parahaemoiyticus are summarized in Table 21
Table 21 Siandard biochemical tesl reactions of V porahaemoyticus
TeSi S Reaction of V parahaem olyaclIs Refe rences Salt tol ance
+
Sali ne TSI agar KIA
No HS and gas production
Simmons Citrnh
Voges-Proskauer
Methyl-Red +
[SO (2007)
Jayasinge et al (2007)
Khan et of (2007)
ISO (2007)
Kim et I (1999)
Abide et til (1999)
Kim 11111 (1999)
Khan et til (2007)
Jayasinge el III (20 (0)
Kim et 0 (2 007)
Note (-) ~ nega live (+) ~ positi ve K ~ alka line ~ aC idic
12
CHAPT ER 3
MATERIALS AND METI IODS
31 Samples collection
Eighty raw vegetable samples were collected week ly from two wet markets (WI amp W2) and
two supennarkets (S 1 amp S2) in Kuching Sarawa k The collected samples consisted o f four
di fferent groups of vegetables which are regularly consumed ra w by the locals These samples
wcr~ rand omly selected within ten weeks -period which is from October 20 10 to January
20 11 Duri ng collection each sample was transferred to clean plastic bags labeled acco rding
to the place they were collected and were analyzed immed iately on arriva l to the labo rat ory
The types of raw vegetable sa mples obtained were as shown in Table 31
Tab le 31 Types o f raw vegetable samples co llected from wet markes and supcmlarkels
Group Loca l nam e English nam e Scienti fi c Home l mple coding No or samples
Soil Kubis Cabbage Brassica v eracea K 20 Roots Lobak Canmiddotot Douclfs carola C ~O
mera h Beans KaCtlO g Four-winged PsophOCaJjJ lls KB cO
bow l bean JeIlagulIolobus Shoot Tauge Bean-I_prout Vigna rad mc T 10 Total 80
13
---------~ I I
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
0 139) V vulnijiclIs and V paralwemolyricus Presently gast roenteitis caused by V
parahaemolyticus is extensively documented due to its increasing outbreaks (Alam el 01
2003 Gwendelynne el 01 2005 Gorny 2006 Harth et 01 2009 Roque et 01 2009)
V parahaemolyriclls is widely present in brackish and marine waters and infection to
human is regula rl y associated with the consumption of contaminated raw or undercooked
scafood (Guoxia ng el 01 2009) Ea rli er studies also demonstrated that V parahoemolylicus
can be easily iso lated from costal water sediments suspended particles and plankton due to
its halophi lic characteristic (Marlina el 01 2007 Zinuneman el 01 2007 Zu lkifli el al
2009 Vimala et 01 2010) However recent foodbome outbreaks throughout the world have
been intensive ly linked to consu mption of fresh fruits vegetables and unpasteurized juices
(Gorny 2006) Most recently Tunu ng et 01 (20 10) had rep0l1ed th at the vegetable samples
from supermarkets and wet markets in Selangor Malaysia were posit ive for pathogeni c
species of V parahaemolylicls Conversely there is no ot her known documented research on
the prevalence of this species in vegetables
In this st udy vegetables from two wet markets and two supem1arkets in Kuching
Sa rawak were sampled for the presence of V parallOemolyliclis The sa moles underwent
selective enrichment with alkaline peptone water (APW) supp lemented with 3 NaCI Then
presumptive V parahaemolYlicus were iso lated using TeES agar and CH ROMagar VibrioT
Following isolat ion gram stai ning together with a series of biochemica l tests were perfonned
to assist in the identification and confirmation of the iso lates
3
- bull i ~
IZ Objectives
This study was underta ken with the fo llowing objecti ves
I To isolate presumptive V parahaemolYlicus from marketed vegetabl es samples
in Kuching Sarawak by using Te BS and CHROMagar VibrioTM
II To confirm th e suspected V parahaemolyficus iso lates from marketed
vegetables samples in Kuching Sarawak by usi ng Gram stain and biochemical
tests
4
- bull i ~
USI KhJtlmal Makumat U umk VMVl~m ItLAYSlA AAAWAK
CHAPTER 2
LITERATURE REVIEW
21 The genus Vibrio
The genus Vibrio belongs to the fami ly Vibrionaceae and it consists of at least 34 recogn ized
species (HPA 2007a) In general Vibrio spp are Gram-negat ive fac ulta tively anaerobic
mOlile curved rods bacteria with a single polar flagellum (FEHD 2005) In surface waters of
the world Vibrios are among the mos t common surface organisms (Adeleye et aI 20 J 0)
They are present in both marine and fresh wa ter habiwts and commonly associa ted with
aquatic animals Vibrio pp such as V choerae V parahaemoylicus V vu nificus V
aginoylieus V mimicus V luvia lis V filmissii V melselmikovii V tollisae and V
dalltsea are common human pathogens (Tison 1999 Fi nkelstein 2000) T hey account for a
significant proportion of human infections as most of these vibrios secrete enterotoxins to
foods water or in the gastrointestinal tract (Nishibuchi and DePao la 2005)
22 Vibrio paralJlell1oytictls
Vibrio parahaemoy licus which is formerly known as Pasteurella parahaeliloytlca is a
non-sporing Gram-negat ive facu ltatively-anaerobic bacterium (Curti s el 01 2003) This
5
- ~
halophilic bacterium inhabits warm estuarine waters world wide (Bilung el al 2005) Thus il
reqlllres salt to survive and appears in high er concentrations during the warmer summer
months
The optimum temperature fo r the grow th of V parahaemolylicus is 37 degC and at Ihis
optimum temperature the bac terium has generation times of8 to 9 minutes in water and 12 [ 0
18 minutes in seafood (Montville and Matthews 2008) Thi s bacterium is the most IKeiy
Vibrio species to be imp licated in food borne diseases although both V vulnificus and V
cholera may also causes food bom e infections (Lawley e l al 2008) Clinical isolates of V
parrhaemolyticus produce beta type hemolysis on blood aga r (Wagatsuma agar) The
hemolysis is ca ll ed the Kanagawa-phenomenon (KP) which is associated with the production
ofa thermo stabl e d irect hemolys inTOH (Boyd 2008) TOH caused damage to the euka ryotic
cells by acting as a pore fonning toxin that alters the ion balance of cells (Boyd 2008)
Figure 21 SEM observa tlOos of V plrh cm)JJ(cu~
Image source Belas amp Colwe ll ( 1982)
6
-- UI
23 Fresh vegetables
Consumpti on of raw vegetables is common throughout the world not onl y owing to Ihe health
benefit but they are thought to be free from disease causing pathogens However recent food
borne outbreaks worldwi de is associ ated to consumption of fresh frui ts uncooked vegetables
and unpasteurized juices (Gorny 2006) Fruits and vegetable especially those that are eaten
raw without peeling can be the vehicles for transmission of a wide range of mi croorganis m
(Erdogru l and Sener 2005)
Based on Haeton and Jones (2008) E coli Salmonella spp and Listeria spp are
commonly present in vegetable samples Meldrum el 01 (2009) report ed that the two recent
large ou tbreaks in the United Kingdom showed signi fi cant health problems that cou ld ari se
from consumption of contaminated salad Another study of the preva lence of pathogens in
vegetables was reported by Little el 01 (2007) in which they found Listeria monocylOgenes in
ready-to-eat mixed salads in the United Kingdom Several other studi es on d ifferent
microorga nism isolated from vegetabl es were also report ed recent ly by local researchers
(Chai el 01 2008 Han el 01 2009 Ponni ah el 0 2009)
In Mala ys ia the presence of V parahoemo yticus has also been repOIt ed in raw
vegetables samples from wet markets and supeLl11arkets in Selangor (Tunung et 0 20 I 0)
This same study discovered that raw vegetabl es such as OenGnthe st l7i(era (Japanese
parsley) and C[cumis sali vus (Cucumber) were contaminated with virulent V
7
parahaemoiYlicus Consequen tly these vegetables could act as a transmission route and
poses risk to consumers after consumption Tunung el 01 (2011) had also demonstrated that
the vegetable samples fro m wet market contained higher levels of V paraiJaemolylicls as
compared w ith supennarkets The research suggested that there are many factors that can
contri bu te to the distribution of V parahaemoiylicus such as temperature holding ti me as
well as hygiene and sanitation practice (Tunung el aI 2011) However there is no known
documented research on V parahaemolYlicus in raw vegetables in Sarawak
24 Clinical characteristic of V parahaelllolyliclis infections
II parahaemoiyticus causes three distinct illness syndromes which are gastroenteritis wound
or soft tissues infections and septicemia (Sillljee 2007) The most freq uen t syndrome is
gastroeuteritis The symptoms include dianhea with abdomina l cramps nausea vo miting
headache and low-grade fever (S tarlin 2005 WilU1 and Konema n 2006 Montville and
Matthew 2008) Sometimes the diarrhea is bloody w ith stools described as meat washed
since tl1e stool is reddish and watery (Qadri el aI 2009)
As for wound or tis sue in fections this coudition occurs by the exposure of broken skin
or wound to warm seawater and seafood dripp ings (Ralph and Currie 2007 Simjee 2007)
In severe cases serious wound infection will resu lt in necrotizing fasc iiti s (Ralph and Curri e
2007) Although less common in V paraiwemolyliclIs infection septicemia is characteri7cd
by fever or hypotension with the isolation of V parahaemolyticus in the blood Septicemia
typicall y occurs in person with underlying health problems such as liver disease diabetes
cancer or other immune disorder (Simjee 2007) V parahaemoyticlis can even ca use fala l
septicemia in an immune-compromised hosts
The symptoms usuall y occur on the average of 15 hours but can began as early as 4
hours and as late as 36 hours after exposure and this will continue up to 3 days (Barceloux
200S) However these illnesses are usua lly categorized as a short illnesses and no prolonged
enect will take place after recovery (Wright et al 2009)
25 Isolation of V parahael1lolyliclIs
For the isolation of V parahaemoyticus an initial emichment using alka line peptone wat er
(APW) with pH S5 would be useful as this medium supports the growth uf viable ce lls
(Simjee 2007) Plati ng of emichment broth onto TCBS agar has been widely used for
selective isolation of V parahaemolytics from foods (Thompson el ai 2006 Oi Pinto et al
201 0 Canizalez -Roman et ai 2011 ) An average of 24 hours incubation at 37 degC is requ ired
fo r typica l V parahaemolylicus to develop colonies that are smooth gree n (s u~rose nega tive)
and 2 to 3 nun in di ameter on TCBS (ISO 2007) However V parahaenlOlytlcus co lonies on
TCBS agar are difficult to distinguish vi suall y from other bacterial colonies since they can be
covered by yellow color produced by sucrose-fennenting bacteria (Thompson el 01 2006
Rambach 2007)
9
_ ------~-- i ~~
Another widely used selective medium is CHROMagar VibrioT On this medium V
pllrahaemoiy liclis will developed mau ve color colonies (Kudo et aI 2001 Oi Pi nto 2010
Canizalez-Roman 2011) This agar medium contains substrates for beta-galactosidase which
was developed specifically to differentiate V parahaemolytics from ot her bacteria by us ing a
chromogenic su bstrate instead of sugar fermentation in traditional growth med ia such as
TCBS (Hara-Kudo et 01 2001) Therefore it is more sensitiv e and accurate for identi fying V
porahllemolyticlIs in food samples rather than the use of Te ES (Oi Pinto 2010 Canizalez-
Roman 20 II)
26 Identification through Gram-stain and biochemical tests
V parahaemoiyticus is a Gram-negative bacterium th us this organi sm stained pink or red
after Gram staining (HPA 20073) Under microscopic observation a typical V
pllrahaemolyticus appeared as rod characterist ica lly curved or comma-shoped The bacterium
is also positi ve for motility under microscopic observations (ISO 2007 BPA 2007a)
V parahaemolylieus ca n be identified through a seri es of biochemical tests According
to Khan et 01 (2007) V para haemolyticus can be distinct ively differentia ted fro m the other
Vibrio spp by performing sa lt tolerance and acid fro m femlen tation tests A typical V
parahaemoiYlicus co lonies would not grow in AFW without th e presence of at least 3 of sali
10
~-------
but it can only endure the presence of salt up to 8 (ISO 2007 Khan el aI 2007) In the
other hand the fermentation test for acid production is the most important test to differentiate
between V parahaemolyliclIs and V vulnificus species (Khan el aI 2007) Jayasinge et ai
(2010) even suggested that the combination of salt toleranc e test Voges-Proskauer test and
ONPG test can provide a strai ghtforward differentiation of V parahaemoly liclis from the
other Vibrionaceae species (Figure 31)
Citrate test screens the ab ilit y of bacterial isolates to utilize ci trate as its carbon and
energy source (MacFaddin 2000) This test is a very important test to identify gram-negative
pathogens (Tang el aI 1998) The MR-VP medium which was used for Met hyl red and
Voges Proskauer test contains glucose and peptone Methyl red test screens for the bacterial
metaboli sm pathways for glucose fermentation while the Voges-Proskauer tes t is an assay for
organisms which ferment glucose with only one fermentation product usuall y acetic acid
The TS[ test is another useful test tha t can distinguish V parahaemolyticus from other
bacteria (ISO 2007) The TSJ agar med ium contains peptone fenous sulfate lactose sucrose
and a small amount of glucose (dex trose) The pH indi cator is phenol red This medium
differentiates bacteria based on their ability to fennent glucose lactose ando r sucrose and on
their abi lity to reduce sulfur to hydrogen sulfide (H S) The T SJ test also haj the abi lity to
determine the production of H]S (Kim et aI 1999)
In additi on other bi ochemica l tests such as oxidase lysine decarboxylase om ithi ne
decarboxylase ca talase and indole can also be used for the purpose of identifying V
II
-- ---- - ----------- - ~i
parahaemoyticus iso lates (ISO 2007 Jayasinghe et ai 2007 Khan et aI 2007) The
biochemical reactions o f V parahaemoiyticus are summarized in Table 21
Table 21 Siandard biochemical tesl reactions of V porahaemoyticus
TeSi S Reaction of V parahaem olyaclIs Refe rences Salt tol ance
+
Sali ne TSI agar KIA
No HS and gas production
Simmons Citrnh
Voges-Proskauer
Methyl-Red +
[SO (2007)
Jayasinge et al (2007)
Khan et of (2007)
ISO (2007)
Kim et I (1999)
Abide et til (1999)
Kim 11111 (1999)
Khan et til (2007)
Jayasinge el III (20 (0)
Kim et 0 (2 007)
Note (-) ~ nega live (+) ~ positi ve K ~ alka line ~ aC idic
12
CHAPT ER 3
MATERIALS AND METI IODS
31 Samples collection
Eighty raw vegetable samples were collected week ly from two wet markets (WI amp W2) and
two supennarkets (S 1 amp S2) in Kuching Sarawa k The collected samples consisted o f four
di fferent groups of vegetables which are regularly consumed ra w by the locals These samples
wcr~ rand omly selected within ten weeks -period which is from October 20 10 to January
20 11 Duri ng collection each sample was transferred to clean plastic bags labeled acco rding
to the place they were collected and were analyzed immed iately on arriva l to the labo rat ory
The types of raw vegetable sa mples obtained were as shown in Table 31
Tab le 31 Types o f raw vegetable samples co llected from wet markes and supcmlarkels
Group Loca l nam e English nam e Scienti fi c Home l mple coding No or samples
Soil Kubis Cabbage Brassica v eracea K 20 Roots Lobak Canmiddotot Douclfs carola C ~O
mera h Beans KaCtlO g Four-winged PsophOCaJjJ lls KB cO
bow l bean JeIlagulIolobus Shoot Tauge Bean-I_prout Vigna rad mc T 10 Total 80
13
---------~ I I
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
IZ Objectives
This study was underta ken with the fo llowing objecti ves
I To isolate presumptive V parahaemolYlicus from marketed vegetabl es samples
in Kuching Sarawak by using Te BS and CHROMagar VibrioTM
II To confirm th e suspected V parahaemolyficus iso lates from marketed
vegetables samples in Kuching Sarawak by usi ng Gram stain and biochemical
tests
4
- bull i ~
USI KhJtlmal Makumat U umk VMVl~m ItLAYSlA AAAWAK
CHAPTER 2
LITERATURE REVIEW
21 The genus Vibrio
The genus Vibrio belongs to the fami ly Vibrionaceae and it consists of at least 34 recogn ized
species (HPA 2007a) In general Vibrio spp are Gram-negat ive fac ulta tively anaerobic
mOlile curved rods bacteria with a single polar flagellum (FEHD 2005) In surface waters of
the world Vibrios are among the mos t common surface organisms (Adeleye et aI 20 J 0)
They are present in both marine and fresh wa ter habiwts and commonly associa ted with
aquatic animals Vibrio pp such as V choerae V parahaemoylicus V vu nificus V
aginoylieus V mimicus V luvia lis V filmissii V melselmikovii V tollisae and V
dalltsea are common human pathogens (Tison 1999 Fi nkelstein 2000) T hey account for a
significant proportion of human infections as most of these vibrios secrete enterotoxins to
foods water or in the gastrointestinal tract (Nishibuchi and DePao la 2005)
22 Vibrio paralJlell1oytictls
Vibrio parahaemoy licus which is formerly known as Pasteurella parahaeliloytlca is a
non-sporing Gram-negat ive facu ltatively-anaerobic bacterium (Curti s el 01 2003) This
5
- ~
halophilic bacterium inhabits warm estuarine waters world wide (Bilung el al 2005) Thus il
reqlllres salt to survive and appears in high er concentrations during the warmer summer
months
The optimum temperature fo r the grow th of V parahaemolylicus is 37 degC and at Ihis
optimum temperature the bac terium has generation times of8 to 9 minutes in water and 12 [ 0
18 minutes in seafood (Montville and Matthews 2008) Thi s bacterium is the most IKeiy
Vibrio species to be imp licated in food borne diseases although both V vulnificus and V
cholera may also causes food bom e infections (Lawley e l al 2008) Clinical isolates of V
parrhaemolyticus produce beta type hemolysis on blood aga r (Wagatsuma agar) The
hemolysis is ca ll ed the Kanagawa-phenomenon (KP) which is associated with the production
ofa thermo stabl e d irect hemolys inTOH (Boyd 2008) TOH caused damage to the euka ryotic
cells by acting as a pore fonning toxin that alters the ion balance of cells (Boyd 2008)
Figure 21 SEM observa tlOos of V plrh cm)JJ(cu~
Image source Belas amp Colwe ll ( 1982)
6
-- UI
23 Fresh vegetables
Consumpti on of raw vegetables is common throughout the world not onl y owing to Ihe health
benefit but they are thought to be free from disease causing pathogens However recent food
borne outbreaks worldwi de is associ ated to consumption of fresh frui ts uncooked vegetables
and unpasteurized juices (Gorny 2006) Fruits and vegetable especially those that are eaten
raw without peeling can be the vehicles for transmission of a wide range of mi croorganis m
(Erdogru l and Sener 2005)
Based on Haeton and Jones (2008) E coli Salmonella spp and Listeria spp are
commonly present in vegetable samples Meldrum el 01 (2009) report ed that the two recent
large ou tbreaks in the United Kingdom showed signi fi cant health problems that cou ld ari se
from consumption of contaminated salad Another study of the preva lence of pathogens in
vegetables was reported by Little el 01 (2007) in which they found Listeria monocylOgenes in
ready-to-eat mixed salads in the United Kingdom Several other studi es on d ifferent
microorga nism isolated from vegetabl es were also report ed recent ly by local researchers
(Chai el 01 2008 Han el 01 2009 Ponni ah el 0 2009)
In Mala ys ia the presence of V parahoemo yticus has also been repOIt ed in raw
vegetables samples from wet markets and supeLl11arkets in Selangor (Tunung et 0 20 I 0)
This same study discovered that raw vegetabl es such as OenGnthe st l7i(era (Japanese
parsley) and C[cumis sali vus (Cucumber) were contaminated with virulent V
7
parahaemoiYlicus Consequen tly these vegetables could act as a transmission route and
poses risk to consumers after consumption Tunung el 01 (2011) had also demonstrated that
the vegetable samples fro m wet market contained higher levels of V paraiJaemolylicls as
compared w ith supennarkets The research suggested that there are many factors that can
contri bu te to the distribution of V parahaemoiylicus such as temperature holding ti me as
well as hygiene and sanitation practice (Tunung el aI 2011) However there is no known
documented research on V parahaemolYlicus in raw vegetables in Sarawak
24 Clinical characteristic of V parahaelllolyliclis infections
II parahaemoiyticus causes three distinct illness syndromes which are gastroenteritis wound
or soft tissues infections and septicemia (Sillljee 2007) The most freq uen t syndrome is
gastroeuteritis The symptoms include dianhea with abdomina l cramps nausea vo miting
headache and low-grade fever (S tarlin 2005 WilU1 and Konema n 2006 Montville and
Matthew 2008) Sometimes the diarrhea is bloody w ith stools described as meat washed
since tl1e stool is reddish and watery (Qadri el aI 2009)
As for wound or tis sue in fections this coudition occurs by the exposure of broken skin
or wound to warm seawater and seafood dripp ings (Ralph and Currie 2007 Simjee 2007)
In severe cases serious wound infection will resu lt in necrotizing fasc iiti s (Ralph and Curri e
2007) Although less common in V paraiwemolyliclIs infection septicemia is characteri7cd
by fever or hypotension with the isolation of V parahaemolyticus in the blood Septicemia
typicall y occurs in person with underlying health problems such as liver disease diabetes
cancer or other immune disorder (Simjee 2007) V parahaemoyticlis can even ca use fala l
septicemia in an immune-compromised hosts
The symptoms usuall y occur on the average of 15 hours but can began as early as 4
hours and as late as 36 hours after exposure and this will continue up to 3 days (Barceloux
200S) However these illnesses are usua lly categorized as a short illnesses and no prolonged
enect will take place after recovery (Wright et al 2009)
25 Isolation of V parahael1lolyliclIs
For the isolation of V parahaemoyticus an initial emichment using alka line peptone wat er
(APW) with pH S5 would be useful as this medium supports the growth uf viable ce lls
(Simjee 2007) Plati ng of emichment broth onto TCBS agar has been widely used for
selective isolation of V parahaemolytics from foods (Thompson el ai 2006 Oi Pinto et al
201 0 Canizalez -Roman et ai 2011 ) An average of 24 hours incubation at 37 degC is requ ired
fo r typica l V parahaemolylicus to develop colonies that are smooth gree n (s u~rose nega tive)
and 2 to 3 nun in di ameter on TCBS (ISO 2007) However V parahaenlOlytlcus co lonies on
TCBS agar are difficult to distinguish vi suall y from other bacterial colonies since they can be
covered by yellow color produced by sucrose-fennenting bacteria (Thompson el 01 2006
Rambach 2007)
9
_ ------~-- i ~~
Another widely used selective medium is CHROMagar VibrioT On this medium V
pllrahaemoiy liclis will developed mau ve color colonies (Kudo et aI 2001 Oi Pi nto 2010
Canizalez-Roman 2011) This agar medium contains substrates for beta-galactosidase which
was developed specifically to differentiate V parahaemolytics from ot her bacteria by us ing a
chromogenic su bstrate instead of sugar fermentation in traditional growth med ia such as
TCBS (Hara-Kudo et 01 2001) Therefore it is more sensitiv e and accurate for identi fying V
porahllemolyticlIs in food samples rather than the use of Te ES (Oi Pinto 2010 Canizalez-
Roman 20 II)
26 Identification through Gram-stain and biochemical tests
V parahaemoiyticus is a Gram-negative bacterium th us this organi sm stained pink or red
after Gram staining (HPA 20073) Under microscopic observation a typical V
pllrahaemolyticus appeared as rod characterist ica lly curved or comma-shoped The bacterium
is also positi ve for motility under microscopic observations (ISO 2007 BPA 2007a)
V parahaemolylieus ca n be identified through a seri es of biochemical tests According
to Khan et 01 (2007) V para haemolyticus can be distinct ively differentia ted fro m the other
Vibrio spp by performing sa lt tolerance and acid fro m femlen tation tests A typical V
parahaemoiYlicus co lonies would not grow in AFW without th e presence of at least 3 of sali
10
~-------
but it can only endure the presence of salt up to 8 (ISO 2007 Khan el aI 2007) In the
other hand the fermentation test for acid production is the most important test to differentiate
between V parahaemolyliclIs and V vulnificus species (Khan el aI 2007) Jayasinge et ai
(2010) even suggested that the combination of salt toleranc e test Voges-Proskauer test and
ONPG test can provide a strai ghtforward differentiation of V parahaemoly liclis from the
other Vibrionaceae species (Figure 31)
Citrate test screens the ab ilit y of bacterial isolates to utilize ci trate as its carbon and
energy source (MacFaddin 2000) This test is a very important test to identify gram-negative
pathogens (Tang el aI 1998) The MR-VP medium which was used for Met hyl red and
Voges Proskauer test contains glucose and peptone Methyl red test screens for the bacterial
metaboli sm pathways for glucose fermentation while the Voges-Proskauer tes t is an assay for
organisms which ferment glucose with only one fermentation product usuall y acetic acid
The TS[ test is another useful test tha t can distinguish V parahaemolyticus from other
bacteria (ISO 2007) The TSJ agar med ium contains peptone fenous sulfate lactose sucrose
and a small amount of glucose (dex trose) The pH indi cator is phenol red This medium
differentiates bacteria based on their ability to fennent glucose lactose ando r sucrose and on
their abi lity to reduce sulfur to hydrogen sulfide (H S) The T SJ test also haj the abi lity to
determine the production of H]S (Kim et aI 1999)
In additi on other bi ochemica l tests such as oxidase lysine decarboxylase om ithi ne
decarboxylase ca talase and indole can also be used for the purpose of identifying V
II
-- ---- - ----------- - ~i
parahaemoyticus iso lates (ISO 2007 Jayasinghe et ai 2007 Khan et aI 2007) The
biochemical reactions o f V parahaemoiyticus are summarized in Table 21
Table 21 Siandard biochemical tesl reactions of V porahaemoyticus
TeSi S Reaction of V parahaem olyaclIs Refe rences Salt tol ance
+
Sali ne TSI agar KIA
No HS and gas production
Simmons Citrnh
Voges-Proskauer
Methyl-Red +
[SO (2007)
Jayasinge et al (2007)
Khan et of (2007)
ISO (2007)
Kim et I (1999)
Abide et til (1999)
Kim 11111 (1999)
Khan et til (2007)
Jayasinge el III (20 (0)
Kim et 0 (2 007)
Note (-) ~ nega live (+) ~ positi ve K ~ alka line ~ aC idic
12
CHAPT ER 3
MATERIALS AND METI IODS
31 Samples collection
Eighty raw vegetable samples were collected week ly from two wet markets (WI amp W2) and
two supennarkets (S 1 amp S2) in Kuching Sarawa k The collected samples consisted o f four
di fferent groups of vegetables which are regularly consumed ra w by the locals These samples
wcr~ rand omly selected within ten weeks -period which is from October 20 10 to January
20 11 Duri ng collection each sample was transferred to clean plastic bags labeled acco rding
to the place they were collected and were analyzed immed iately on arriva l to the labo rat ory
The types of raw vegetable sa mples obtained were as shown in Table 31
Tab le 31 Types o f raw vegetable samples co llected from wet markes and supcmlarkels
Group Loca l nam e English nam e Scienti fi c Home l mple coding No or samples
Soil Kubis Cabbage Brassica v eracea K 20 Roots Lobak Canmiddotot Douclfs carola C ~O
mera h Beans KaCtlO g Four-winged PsophOCaJjJ lls KB cO
bow l bean JeIlagulIolobus Shoot Tauge Bean-I_prout Vigna rad mc T 10 Total 80
13
---------~ I I
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
USI KhJtlmal Makumat U umk VMVl~m ItLAYSlA AAAWAK
CHAPTER 2
LITERATURE REVIEW
21 The genus Vibrio
The genus Vibrio belongs to the fami ly Vibrionaceae and it consists of at least 34 recogn ized
species (HPA 2007a) In general Vibrio spp are Gram-negat ive fac ulta tively anaerobic
mOlile curved rods bacteria with a single polar flagellum (FEHD 2005) In surface waters of
the world Vibrios are among the mos t common surface organisms (Adeleye et aI 20 J 0)
They are present in both marine and fresh wa ter habiwts and commonly associa ted with
aquatic animals Vibrio pp such as V choerae V parahaemoylicus V vu nificus V
aginoylieus V mimicus V luvia lis V filmissii V melselmikovii V tollisae and V
dalltsea are common human pathogens (Tison 1999 Fi nkelstein 2000) T hey account for a
significant proportion of human infections as most of these vibrios secrete enterotoxins to
foods water or in the gastrointestinal tract (Nishibuchi and DePao la 2005)
22 Vibrio paralJlell1oytictls
Vibrio parahaemoy licus which is formerly known as Pasteurella parahaeliloytlca is a
non-sporing Gram-negat ive facu ltatively-anaerobic bacterium (Curti s el 01 2003) This
5
- ~
halophilic bacterium inhabits warm estuarine waters world wide (Bilung el al 2005) Thus il
reqlllres salt to survive and appears in high er concentrations during the warmer summer
months
The optimum temperature fo r the grow th of V parahaemolylicus is 37 degC and at Ihis
optimum temperature the bac terium has generation times of8 to 9 minutes in water and 12 [ 0
18 minutes in seafood (Montville and Matthews 2008) Thi s bacterium is the most IKeiy
Vibrio species to be imp licated in food borne diseases although both V vulnificus and V
cholera may also causes food bom e infections (Lawley e l al 2008) Clinical isolates of V
parrhaemolyticus produce beta type hemolysis on blood aga r (Wagatsuma agar) The
hemolysis is ca ll ed the Kanagawa-phenomenon (KP) which is associated with the production
ofa thermo stabl e d irect hemolys inTOH (Boyd 2008) TOH caused damage to the euka ryotic
cells by acting as a pore fonning toxin that alters the ion balance of cells (Boyd 2008)
Figure 21 SEM observa tlOos of V plrh cm)JJ(cu~
Image source Belas amp Colwe ll ( 1982)
6
-- UI
23 Fresh vegetables
Consumpti on of raw vegetables is common throughout the world not onl y owing to Ihe health
benefit but they are thought to be free from disease causing pathogens However recent food
borne outbreaks worldwi de is associ ated to consumption of fresh frui ts uncooked vegetables
and unpasteurized juices (Gorny 2006) Fruits and vegetable especially those that are eaten
raw without peeling can be the vehicles for transmission of a wide range of mi croorganis m
(Erdogru l and Sener 2005)
Based on Haeton and Jones (2008) E coli Salmonella spp and Listeria spp are
commonly present in vegetable samples Meldrum el 01 (2009) report ed that the two recent
large ou tbreaks in the United Kingdom showed signi fi cant health problems that cou ld ari se
from consumption of contaminated salad Another study of the preva lence of pathogens in
vegetables was reported by Little el 01 (2007) in which they found Listeria monocylOgenes in
ready-to-eat mixed salads in the United Kingdom Several other studi es on d ifferent
microorga nism isolated from vegetabl es were also report ed recent ly by local researchers
(Chai el 01 2008 Han el 01 2009 Ponni ah el 0 2009)
In Mala ys ia the presence of V parahoemo yticus has also been repOIt ed in raw
vegetables samples from wet markets and supeLl11arkets in Selangor (Tunung et 0 20 I 0)
This same study discovered that raw vegetabl es such as OenGnthe st l7i(era (Japanese
parsley) and C[cumis sali vus (Cucumber) were contaminated with virulent V
7
parahaemoiYlicus Consequen tly these vegetables could act as a transmission route and
poses risk to consumers after consumption Tunung el 01 (2011) had also demonstrated that
the vegetable samples fro m wet market contained higher levels of V paraiJaemolylicls as
compared w ith supennarkets The research suggested that there are many factors that can
contri bu te to the distribution of V parahaemoiylicus such as temperature holding ti me as
well as hygiene and sanitation practice (Tunung el aI 2011) However there is no known
documented research on V parahaemolYlicus in raw vegetables in Sarawak
24 Clinical characteristic of V parahaelllolyliclis infections
II parahaemoiyticus causes three distinct illness syndromes which are gastroenteritis wound
or soft tissues infections and septicemia (Sillljee 2007) The most freq uen t syndrome is
gastroeuteritis The symptoms include dianhea with abdomina l cramps nausea vo miting
headache and low-grade fever (S tarlin 2005 WilU1 and Konema n 2006 Montville and
Matthew 2008) Sometimes the diarrhea is bloody w ith stools described as meat washed
since tl1e stool is reddish and watery (Qadri el aI 2009)
As for wound or tis sue in fections this coudition occurs by the exposure of broken skin
or wound to warm seawater and seafood dripp ings (Ralph and Currie 2007 Simjee 2007)
In severe cases serious wound infection will resu lt in necrotizing fasc iiti s (Ralph and Curri e
2007) Although less common in V paraiwemolyliclIs infection septicemia is characteri7cd
by fever or hypotension with the isolation of V parahaemolyticus in the blood Septicemia
typicall y occurs in person with underlying health problems such as liver disease diabetes
cancer or other immune disorder (Simjee 2007) V parahaemoyticlis can even ca use fala l
septicemia in an immune-compromised hosts
The symptoms usuall y occur on the average of 15 hours but can began as early as 4
hours and as late as 36 hours after exposure and this will continue up to 3 days (Barceloux
200S) However these illnesses are usua lly categorized as a short illnesses and no prolonged
enect will take place after recovery (Wright et al 2009)
25 Isolation of V parahael1lolyliclIs
For the isolation of V parahaemoyticus an initial emichment using alka line peptone wat er
(APW) with pH S5 would be useful as this medium supports the growth uf viable ce lls
(Simjee 2007) Plati ng of emichment broth onto TCBS agar has been widely used for
selective isolation of V parahaemolytics from foods (Thompson el ai 2006 Oi Pinto et al
201 0 Canizalez -Roman et ai 2011 ) An average of 24 hours incubation at 37 degC is requ ired
fo r typica l V parahaemolylicus to develop colonies that are smooth gree n (s u~rose nega tive)
and 2 to 3 nun in di ameter on TCBS (ISO 2007) However V parahaenlOlytlcus co lonies on
TCBS agar are difficult to distinguish vi suall y from other bacterial colonies since they can be
covered by yellow color produced by sucrose-fennenting bacteria (Thompson el 01 2006
Rambach 2007)
9
_ ------~-- i ~~
Another widely used selective medium is CHROMagar VibrioT On this medium V
pllrahaemoiy liclis will developed mau ve color colonies (Kudo et aI 2001 Oi Pi nto 2010
Canizalez-Roman 2011) This agar medium contains substrates for beta-galactosidase which
was developed specifically to differentiate V parahaemolytics from ot her bacteria by us ing a
chromogenic su bstrate instead of sugar fermentation in traditional growth med ia such as
TCBS (Hara-Kudo et 01 2001) Therefore it is more sensitiv e and accurate for identi fying V
porahllemolyticlIs in food samples rather than the use of Te ES (Oi Pinto 2010 Canizalez-
Roman 20 II)
26 Identification through Gram-stain and biochemical tests
V parahaemoiyticus is a Gram-negative bacterium th us this organi sm stained pink or red
after Gram staining (HPA 20073) Under microscopic observation a typical V
pllrahaemolyticus appeared as rod characterist ica lly curved or comma-shoped The bacterium
is also positi ve for motility under microscopic observations (ISO 2007 BPA 2007a)
V parahaemolylieus ca n be identified through a seri es of biochemical tests According
to Khan et 01 (2007) V para haemolyticus can be distinct ively differentia ted fro m the other
Vibrio spp by performing sa lt tolerance and acid fro m femlen tation tests A typical V
parahaemoiYlicus co lonies would not grow in AFW without th e presence of at least 3 of sali
10
~-------
but it can only endure the presence of salt up to 8 (ISO 2007 Khan el aI 2007) In the
other hand the fermentation test for acid production is the most important test to differentiate
between V parahaemolyliclIs and V vulnificus species (Khan el aI 2007) Jayasinge et ai
(2010) even suggested that the combination of salt toleranc e test Voges-Proskauer test and
ONPG test can provide a strai ghtforward differentiation of V parahaemoly liclis from the
other Vibrionaceae species (Figure 31)
Citrate test screens the ab ilit y of bacterial isolates to utilize ci trate as its carbon and
energy source (MacFaddin 2000) This test is a very important test to identify gram-negative
pathogens (Tang el aI 1998) The MR-VP medium which was used for Met hyl red and
Voges Proskauer test contains glucose and peptone Methyl red test screens for the bacterial
metaboli sm pathways for glucose fermentation while the Voges-Proskauer tes t is an assay for
organisms which ferment glucose with only one fermentation product usuall y acetic acid
The TS[ test is another useful test tha t can distinguish V parahaemolyticus from other
bacteria (ISO 2007) The TSJ agar med ium contains peptone fenous sulfate lactose sucrose
and a small amount of glucose (dex trose) The pH indi cator is phenol red This medium
differentiates bacteria based on their ability to fennent glucose lactose ando r sucrose and on
their abi lity to reduce sulfur to hydrogen sulfide (H S) The T SJ test also haj the abi lity to
determine the production of H]S (Kim et aI 1999)
In additi on other bi ochemica l tests such as oxidase lysine decarboxylase om ithi ne
decarboxylase ca talase and indole can also be used for the purpose of identifying V
II
-- ---- - ----------- - ~i
parahaemoyticus iso lates (ISO 2007 Jayasinghe et ai 2007 Khan et aI 2007) The
biochemical reactions o f V parahaemoiyticus are summarized in Table 21
Table 21 Siandard biochemical tesl reactions of V porahaemoyticus
TeSi S Reaction of V parahaem olyaclIs Refe rences Salt tol ance
+
Sali ne TSI agar KIA
No HS and gas production
Simmons Citrnh
Voges-Proskauer
Methyl-Red +
[SO (2007)
Jayasinge et al (2007)
Khan et of (2007)
ISO (2007)
Kim et I (1999)
Abide et til (1999)
Kim 11111 (1999)
Khan et til (2007)
Jayasinge el III (20 (0)
Kim et 0 (2 007)
Note (-) ~ nega live (+) ~ positi ve K ~ alka line ~ aC idic
12
CHAPT ER 3
MATERIALS AND METI IODS
31 Samples collection
Eighty raw vegetable samples were collected week ly from two wet markets (WI amp W2) and
two supennarkets (S 1 amp S2) in Kuching Sarawa k The collected samples consisted o f four
di fferent groups of vegetables which are regularly consumed ra w by the locals These samples
wcr~ rand omly selected within ten weeks -period which is from October 20 10 to January
20 11 Duri ng collection each sample was transferred to clean plastic bags labeled acco rding
to the place they were collected and were analyzed immed iately on arriva l to the labo rat ory
The types of raw vegetable sa mples obtained were as shown in Table 31
Tab le 31 Types o f raw vegetable samples co llected from wet markes and supcmlarkels
Group Loca l nam e English nam e Scienti fi c Home l mple coding No or samples
Soil Kubis Cabbage Brassica v eracea K 20 Roots Lobak Canmiddotot Douclfs carola C ~O
mera h Beans KaCtlO g Four-winged PsophOCaJjJ lls KB cO
bow l bean JeIlagulIolobus Shoot Tauge Bean-I_prout Vigna rad mc T 10 Total 80
13
---------~ I I
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
halophilic bacterium inhabits warm estuarine waters world wide (Bilung el al 2005) Thus il
reqlllres salt to survive and appears in high er concentrations during the warmer summer
months
The optimum temperature fo r the grow th of V parahaemolylicus is 37 degC and at Ihis
optimum temperature the bac terium has generation times of8 to 9 minutes in water and 12 [ 0
18 minutes in seafood (Montville and Matthews 2008) Thi s bacterium is the most IKeiy
Vibrio species to be imp licated in food borne diseases although both V vulnificus and V
cholera may also causes food bom e infections (Lawley e l al 2008) Clinical isolates of V
parrhaemolyticus produce beta type hemolysis on blood aga r (Wagatsuma agar) The
hemolysis is ca ll ed the Kanagawa-phenomenon (KP) which is associated with the production
ofa thermo stabl e d irect hemolys inTOH (Boyd 2008) TOH caused damage to the euka ryotic
cells by acting as a pore fonning toxin that alters the ion balance of cells (Boyd 2008)
Figure 21 SEM observa tlOos of V plrh cm)JJ(cu~
Image source Belas amp Colwe ll ( 1982)
6
-- UI
23 Fresh vegetables
Consumpti on of raw vegetables is common throughout the world not onl y owing to Ihe health
benefit but they are thought to be free from disease causing pathogens However recent food
borne outbreaks worldwi de is associ ated to consumption of fresh frui ts uncooked vegetables
and unpasteurized juices (Gorny 2006) Fruits and vegetable especially those that are eaten
raw without peeling can be the vehicles for transmission of a wide range of mi croorganis m
(Erdogru l and Sener 2005)
Based on Haeton and Jones (2008) E coli Salmonella spp and Listeria spp are
commonly present in vegetable samples Meldrum el 01 (2009) report ed that the two recent
large ou tbreaks in the United Kingdom showed signi fi cant health problems that cou ld ari se
from consumption of contaminated salad Another study of the preva lence of pathogens in
vegetables was reported by Little el 01 (2007) in which they found Listeria monocylOgenes in
ready-to-eat mixed salads in the United Kingdom Several other studi es on d ifferent
microorga nism isolated from vegetabl es were also report ed recent ly by local researchers
(Chai el 01 2008 Han el 01 2009 Ponni ah el 0 2009)
In Mala ys ia the presence of V parahoemo yticus has also been repOIt ed in raw
vegetables samples from wet markets and supeLl11arkets in Selangor (Tunung et 0 20 I 0)
This same study discovered that raw vegetabl es such as OenGnthe st l7i(era (Japanese
parsley) and C[cumis sali vus (Cucumber) were contaminated with virulent V
7
parahaemoiYlicus Consequen tly these vegetables could act as a transmission route and
poses risk to consumers after consumption Tunung el 01 (2011) had also demonstrated that
the vegetable samples fro m wet market contained higher levels of V paraiJaemolylicls as
compared w ith supennarkets The research suggested that there are many factors that can
contri bu te to the distribution of V parahaemoiylicus such as temperature holding ti me as
well as hygiene and sanitation practice (Tunung el aI 2011) However there is no known
documented research on V parahaemolYlicus in raw vegetables in Sarawak
24 Clinical characteristic of V parahaelllolyliclis infections
II parahaemoiyticus causes three distinct illness syndromes which are gastroenteritis wound
or soft tissues infections and septicemia (Sillljee 2007) The most freq uen t syndrome is
gastroeuteritis The symptoms include dianhea with abdomina l cramps nausea vo miting
headache and low-grade fever (S tarlin 2005 WilU1 and Konema n 2006 Montville and
Matthew 2008) Sometimes the diarrhea is bloody w ith stools described as meat washed
since tl1e stool is reddish and watery (Qadri el aI 2009)
As for wound or tis sue in fections this coudition occurs by the exposure of broken skin
or wound to warm seawater and seafood dripp ings (Ralph and Currie 2007 Simjee 2007)
In severe cases serious wound infection will resu lt in necrotizing fasc iiti s (Ralph and Curri e
2007) Although less common in V paraiwemolyliclIs infection septicemia is characteri7cd
by fever or hypotension with the isolation of V parahaemolyticus in the blood Septicemia
typicall y occurs in person with underlying health problems such as liver disease diabetes
cancer or other immune disorder (Simjee 2007) V parahaemoyticlis can even ca use fala l
septicemia in an immune-compromised hosts
The symptoms usuall y occur on the average of 15 hours but can began as early as 4
hours and as late as 36 hours after exposure and this will continue up to 3 days (Barceloux
200S) However these illnesses are usua lly categorized as a short illnesses and no prolonged
enect will take place after recovery (Wright et al 2009)
25 Isolation of V parahael1lolyliclIs
For the isolation of V parahaemoyticus an initial emichment using alka line peptone wat er
(APW) with pH S5 would be useful as this medium supports the growth uf viable ce lls
(Simjee 2007) Plati ng of emichment broth onto TCBS agar has been widely used for
selective isolation of V parahaemolytics from foods (Thompson el ai 2006 Oi Pinto et al
201 0 Canizalez -Roman et ai 2011 ) An average of 24 hours incubation at 37 degC is requ ired
fo r typica l V parahaemolylicus to develop colonies that are smooth gree n (s u~rose nega tive)
and 2 to 3 nun in di ameter on TCBS (ISO 2007) However V parahaenlOlytlcus co lonies on
TCBS agar are difficult to distinguish vi suall y from other bacterial colonies since they can be
covered by yellow color produced by sucrose-fennenting bacteria (Thompson el 01 2006
Rambach 2007)
9
_ ------~-- i ~~
Another widely used selective medium is CHROMagar VibrioT On this medium V
pllrahaemoiy liclis will developed mau ve color colonies (Kudo et aI 2001 Oi Pi nto 2010
Canizalez-Roman 2011) This agar medium contains substrates for beta-galactosidase which
was developed specifically to differentiate V parahaemolytics from ot her bacteria by us ing a
chromogenic su bstrate instead of sugar fermentation in traditional growth med ia such as
TCBS (Hara-Kudo et 01 2001) Therefore it is more sensitiv e and accurate for identi fying V
porahllemolyticlIs in food samples rather than the use of Te ES (Oi Pinto 2010 Canizalez-
Roman 20 II)
26 Identification through Gram-stain and biochemical tests
V parahaemoiyticus is a Gram-negative bacterium th us this organi sm stained pink or red
after Gram staining (HPA 20073) Under microscopic observation a typical V
pllrahaemolyticus appeared as rod characterist ica lly curved or comma-shoped The bacterium
is also positi ve for motility under microscopic observations (ISO 2007 BPA 2007a)
V parahaemolylieus ca n be identified through a seri es of biochemical tests According
to Khan et 01 (2007) V para haemolyticus can be distinct ively differentia ted fro m the other
Vibrio spp by performing sa lt tolerance and acid fro m femlen tation tests A typical V
parahaemoiYlicus co lonies would not grow in AFW without th e presence of at least 3 of sali
10
~-------
but it can only endure the presence of salt up to 8 (ISO 2007 Khan el aI 2007) In the
other hand the fermentation test for acid production is the most important test to differentiate
between V parahaemolyliclIs and V vulnificus species (Khan el aI 2007) Jayasinge et ai
(2010) even suggested that the combination of salt toleranc e test Voges-Proskauer test and
ONPG test can provide a strai ghtforward differentiation of V parahaemoly liclis from the
other Vibrionaceae species (Figure 31)
Citrate test screens the ab ilit y of bacterial isolates to utilize ci trate as its carbon and
energy source (MacFaddin 2000) This test is a very important test to identify gram-negative
pathogens (Tang el aI 1998) The MR-VP medium which was used for Met hyl red and
Voges Proskauer test contains glucose and peptone Methyl red test screens for the bacterial
metaboli sm pathways for glucose fermentation while the Voges-Proskauer tes t is an assay for
organisms which ferment glucose with only one fermentation product usuall y acetic acid
The TS[ test is another useful test tha t can distinguish V parahaemolyticus from other
bacteria (ISO 2007) The TSJ agar med ium contains peptone fenous sulfate lactose sucrose
and a small amount of glucose (dex trose) The pH indi cator is phenol red This medium
differentiates bacteria based on their ability to fennent glucose lactose ando r sucrose and on
their abi lity to reduce sulfur to hydrogen sulfide (H S) The T SJ test also haj the abi lity to
determine the production of H]S (Kim et aI 1999)
In additi on other bi ochemica l tests such as oxidase lysine decarboxylase om ithi ne
decarboxylase ca talase and indole can also be used for the purpose of identifying V
II
-- ---- - ----------- - ~i
parahaemoyticus iso lates (ISO 2007 Jayasinghe et ai 2007 Khan et aI 2007) The
biochemical reactions o f V parahaemoiyticus are summarized in Table 21
Table 21 Siandard biochemical tesl reactions of V porahaemoyticus
TeSi S Reaction of V parahaem olyaclIs Refe rences Salt tol ance
+
Sali ne TSI agar KIA
No HS and gas production
Simmons Citrnh
Voges-Proskauer
Methyl-Red +
[SO (2007)
Jayasinge et al (2007)
Khan et of (2007)
ISO (2007)
Kim et I (1999)
Abide et til (1999)
Kim 11111 (1999)
Khan et til (2007)
Jayasinge el III (20 (0)
Kim et 0 (2 007)
Note (-) ~ nega live (+) ~ positi ve K ~ alka line ~ aC idic
12
CHAPT ER 3
MATERIALS AND METI IODS
31 Samples collection
Eighty raw vegetable samples were collected week ly from two wet markets (WI amp W2) and
two supennarkets (S 1 amp S2) in Kuching Sarawa k The collected samples consisted o f four
di fferent groups of vegetables which are regularly consumed ra w by the locals These samples
wcr~ rand omly selected within ten weeks -period which is from October 20 10 to January
20 11 Duri ng collection each sample was transferred to clean plastic bags labeled acco rding
to the place they were collected and were analyzed immed iately on arriva l to the labo rat ory
The types of raw vegetable sa mples obtained were as shown in Table 31
Tab le 31 Types o f raw vegetable samples co llected from wet markes and supcmlarkels
Group Loca l nam e English nam e Scienti fi c Home l mple coding No or samples
Soil Kubis Cabbage Brassica v eracea K 20 Roots Lobak Canmiddotot Douclfs carola C ~O
mera h Beans KaCtlO g Four-winged PsophOCaJjJ lls KB cO
bow l bean JeIlagulIolobus Shoot Tauge Bean-I_prout Vigna rad mc T 10 Total 80
13
---------~ I I
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
23 Fresh vegetables
Consumpti on of raw vegetables is common throughout the world not onl y owing to Ihe health
benefit but they are thought to be free from disease causing pathogens However recent food
borne outbreaks worldwi de is associ ated to consumption of fresh frui ts uncooked vegetables
and unpasteurized juices (Gorny 2006) Fruits and vegetable especially those that are eaten
raw without peeling can be the vehicles for transmission of a wide range of mi croorganis m
(Erdogru l and Sener 2005)
Based on Haeton and Jones (2008) E coli Salmonella spp and Listeria spp are
commonly present in vegetable samples Meldrum el 01 (2009) report ed that the two recent
large ou tbreaks in the United Kingdom showed signi fi cant health problems that cou ld ari se
from consumption of contaminated salad Another study of the preva lence of pathogens in
vegetables was reported by Little el 01 (2007) in which they found Listeria monocylOgenes in
ready-to-eat mixed salads in the United Kingdom Several other studi es on d ifferent
microorga nism isolated from vegetabl es were also report ed recent ly by local researchers
(Chai el 01 2008 Han el 01 2009 Ponni ah el 0 2009)
In Mala ys ia the presence of V parahoemo yticus has also been repOIt ed in raw
vegetables samples from wet markets and supeLl11arkets in Selangor (Tunung et 0 20 I 0)
This same study discovered that raw vegetabl es such as OenGnthe st l7i(era (Japanese
parsley) and C[cumis sali vus (Cucumber) were contaminated with virulent V
7
parahaemoiYlicus Consequen tly these vegetables could act as a transmission route and
poses risk to consumers after consumption Tunung el 01 (2011) had also demonstrated that
the vegetable samples fro m wet market contained higher levels of V paraiJaemolylicls as
compared w ith supennarkets The research suggested that there are many factors that can
contri bu te to the distribution of V parahaemoiylicus such as temperature holding ti me as
well as hygiene and sanitation practice (Tunung el aI 2011) However there is no known
documented research on V parahaemolYlicus in raw vegetables in Sarawak
24 Clinical characteristic of V parahaelllolyliclis infections
II parahaemoiyticus causes three distinct illness syndromes which are gastroenteritis wound
or soft tissues infections and septicemia (Sillljee 2007) The most freq uen t syndrome is
gastroeuteritis The symptoms include dianhea with abdomina l cramps nausea vo miting
headache and low-grade fever (S tarlin 2005 WilU1 and Konema n 2006 Montville and
Matthew 2008) Sometimes the diarrhea is bloody w ith stools described as meat washed
since tl1e stool is reddish and watery (Qadri el aI 2009)
As for wound or tis sue in fections this coudition occurs by the exposure of broken skin
or wound to warm seawater and seafood dripp ings (Ralph and Currie 2007 Simjee 2007)
In severe cases serious wound infection will resu lt in necrotizing fasc iiti s (Ralph and Curri e
2007) Although less common in V paraiwemolyliclIs infection septicemia is characteri7cd
by fever or hypotension with the isolation of V parahaemolyticus in the blood Septicemia
typicall y occurs in person with underlying health problems such as liver disease diabetes
cancer or other immune disorder (Simjee 2007) V parahaemoyticlis can even ca use fala l
septicemia in an immune-compromised hosts
The symptoms usuall y occur on the average of 15 hours but can began as early as 4
hours and as late as 36 hours after exposure and this will continue up to 3 days (Barceloux
200S) However these illnesses are usua lly categorized as a short illnesses and no prolonged
enect will take place after recovery (Wright et al 2009)
25 Isolation of V parahael1lolyliclIs
For the isolation of V parahaemoyticus an initial emichment using alka line peptone wat er
(APW) with pH S5 would be useful as this medium supports the growth uf viable ce lls
(Simjee 2007) Plati ng of emichment broth onto TCBS agar has been widely used for
selective isolation of V parahaemolytics from foods (Thompson el ai 2006 Oi Pinto et al
201 0 Canizalez -Roman et ai 2011 ) An average of 24 hours incubation at 37 degC is requ ired
fo r typica l V parahaemolylicus to develop colonies that are smooth gree n (s u~rose nega tive)
and 2 to 3 nun in di ameter on TCBS (ISO 2007) However V parahaenlOlytlcus co lonies on
TCBS agar are difficult to distinguish vi suall y from other bacterial colonies since they can be
covered by yellow color produced by sucrose-fennenting bacteria (Thompson el 01 2006
Rambach 2007)
9
_ ------~-- i ~~
Another widely used selective medium is CHROMagar VibrioT On this medium V
pllrahaemoiy liclis will developed mau ve color colonies (Kudo et aI 2001 Oi Pi nto 2010
Canizalez-Roman 2011) This agar medium contains substrates for beta-galactosidase which
was developed specifically to differentiate V parahaemolytics from ot her bacteria by us ing a
chromogenic su bstrate instead of sugar fermentation in traditional growth med ia such as
TCBS (Hara-Kudo et 01 2001) Therefore it is more sensitiv e and accurate for identi fying V
porahllemolyticlIs in food samples rather than the use of Te ES (Oi Pinto 2010 Canizalez-
Roman 20 II)
26 Identification through Gram-stain and biochemical tests
V parahaemoiyticus is a Gram-negative bacterium th us this organi sm stained pink or red
after Gram staining (HPA 20073) Under microscopic observation a typical V
pllrahaemolyticus appeared as rod characterist ica lly curved or comma-shoped The bacterium
is also positi ve for motility under microscopic observations (ISO 2007 BPA 2007a)
V parahaemolylieus ca n be identified through a seri es of biochemical tests According
to Khan et 01 (2007) V para haemolyticus can be distinct ively differentia ted fro m the other
Vibrio spp by performing sa lt tolerance and acid fro m femlen tation tests A typical V
parahaemoiYlicus co lonies would not grow in AFW without th e presence of at least 3 of sali
10
~-------
but it can only endure the presence of salt up to 8 (ISO 2007 Khan el aI 2007) In the
other hand the fermentation test for acid production is the most important test to differentiate
between V parahaemolyliclIs and V vulnificus species (Khan el aI 2007) Jayasinge et ai
(2010) even suggested that the combination of salt toleranc e test Voges-Proskauer test and
ONPG test can provide a strai ghtforward differentiation of V parahaemoly liclis from the
other Vibrionaceae species (Figure 31)
Citrate test screens the ab ilit y of bacterial isolates to utilize ci trate as its carbon and
energy source (MacFaddin 2000) This test is a very important test to identify gram-negative
pathogens (Tang el aI 1998) The MR-VP medium which was used for Met hyl red and
Voges Proskauer test contains glucose and peptone Methyl red test screens for the bacterial
metaboli sm pathways for glucose fermentation while the Voges-Proskauer tes t is an assay for
organisms which ferment glucose with only one fermentation product usuall y acetic acid
The TS[ test is another useful test tha t can distinguish V parahaemolyticus from other
bacteria (ISO 2007) The TSJ agar med ium contains peptone fenous sulfate lactose sucrose
and a small amount of glucose (dex trose) The pH indi cator is phenol red This medium
differentiates bacteria based on their ability to fennent glucose lactose ando r sucrose and on
their abi lity to reduce sulfur to hydrogen sulfide (H S) The T SJ test also haj the abi lity to
determine the production of H]S (Kim et aI 1999)
In additi on other bi ochemica l tests such as oxidase lysine decarboxylase om ithi ne
decarboxylase ca talase and indole can also be used for the purpose of identifying V
II
-- ---- - ----------- - ~i
parahaemoyticus iso lates (ISO 2007 Jayasinghe et ai 2007 Khan et aI 2007) The
biochemical reactions o f V parahaemoiyticus are summarized in Table 21
Table 21 Siandard biochemical tesl reactions of V porahaemoyticus
TeSi S Reaction of V parahaem olyaclIs Refe rences Salt tol ance
+
Sali ne TSI agar KIA
No HS and gas production
Simmons Citrnh
Voges-Proskauer
Methyl-Red +
[SO (2007)
Jayasinge et al (2007)
Khan et of (2007)
ISO (2007)
Kim et I (1999)
Abide et til (1999)
Kim 11111 (1999)
Khan et til (2007)
Jayasinge el III (20 (0)
Kim et 0 (2 007)
Note (-) ~ nega live (+) ~ positi ve K ~ alka line ~ aC idic
12
CHAPT ER 3
MATERIALS AND METI IODS
31 Samples collection
Eighty raw vegetable samples were collected week ly from two wet markets (WI amp W2) and
two supennarkets (S 1 amp S2) in Kuching Sarawa k The collected samples consisted o f four
di fferent groups of vegetables which are regularly consumed ra w by the locals These samples
wcr~ rand omly selected within ten weeks -period which is from October 20 10 to January
20 11 Duri ng collection each sample was transferred to clean plastic bags labeled acco rding
to the place they were collected and were analyzed immed iately on arriva l to the labo rat ory
The types of raw vegetable sa mples obtained were as shown in Table 31
Tab le 31 Types o f raw vegetable samples co llected from wet markes and supcmlarkels
Group Loca l nam e English nam e Scienti fi c Home l mple coding No or samples
Soil Kubis Cabbage Brassica v eracea K 20 Roots Lobak Canmiddotot Douclfs carola C ~O
mera h Beans KaCtlO g Four-winged PsophOCaJjJ lls KB cO
bow l bean JeIlagulIolobus Shoot Tauge Bean-I_prout Vigna rad mc T 10 Total 80
13
---------~ I I
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
parahaemoiYlicus Consequen tly these vegetables could act as a transmission route and
poses risk to consumers after consumption Tunung el 01 (2011) had also demonstrated that
the vegetable samples fro m wet market contained higher levels of V paraiJaemolylicls as
compared w ith supennarkets The research suggested that there are many factors that can
contri bu te to the distribution of V parahaemoiylicus such as temperature holding ti me as
well as hygiene and sanitation practice (Tunung el aI 2011) However there is no known
documented research on V parahaemolYlicus in raw vegetables in Sarawak
24 Clinical characteristic of V parahaelllolyliclis infections
II parahaemoiyticus causes three distinct illness syndromes which are gastroenteritis wound
or soft tissues infections and septicemia (Sillljee 2007) The most freq uen t syndrome is
gastroeuteritis The symptoms include dianhea with abdomina l cramps nausea vo miting
headache and low-grade fever (S tarlin 2005 WilU1 and Konema n 2006 Montville and
Matthew 2008) Sometimes the diarrhea is bloody w ith stools described as meat washed
since tl1e stool is reddish and watery (Qadri el aI 2009)
As for wound or tis sue in fections this coudition occurs by the exposure of broken skin
or wound to warm seawater and seafood dripp ings (Ralph and Currie 2007 Simjee 2007)
In severe cases serious wound infection will resu lt in necrotizing fasc iiti s (Ralph and Curri e
2007) Although less common in V paraiwemolyliclIs infection septicemia is characteri7cd
by fever or hypotension with the isolation of V parahaemolyticus in the blood Septicemia
typicall y occurs in person with underlying health problems such as liver disease diabetes
cancer or other immune disorder (Simjee 2007) V parahaemoyticlis can even ca use fala l
septicemia in an immune-compromised hosts
The symptoms usuall y occur on the average of 15 hours but can began as early as 4
hours and as late as 36 hours after exposure and this will continue up to 3 days (Barceloux
200S) However these illnesses are usua lly categorized as a short illnesses and no prolonged
enect will take place after recovery (Wright et al 2009)
25 Isolation of V parahael1lolyliclIs
For the isolation of V parahaemoyticus an initial emichment using alka line peptone wat er
(APW) with pH S5 would be useful as this medium supports the growth uf viable ce lls
(Simjee 2007) Plati ng of emichment broth onto TCBS agar has been widely used for
selective isolation of V parahaemolytics from foods (Thompson el ai 2006 Oi Pinto et al
201 0 Canizalez -Roman et ai 2011 ) An average of 24 hours incubation at 37 degC is requ ired
fo r typica l V parahaemolylicus to develop colonies that are smooth gree n (s u~rose nega tive)
and 2 to 3 nun in di ameter on TCBS (ISO 2007) However V parahaenlOlytlcus co lonies on
TCBS agar are difficult to distinguish vi suall y from other bacterial colonies since they can be
covered by yellow color produced by sucrose-fennenting bacteria (Thompson el 01 2006
Rambach 2007)
9
_ ------~-- i ~~
Another widely used selective medium is CHROMagar VibrioT On this medium V
pllrahaemoiy liclis will developed mau ve color colonies (Kudo et aI 2001 Oi Pi nto 2010
Canizalez-Roman 2011) This agar medium contains substrates for beta-galactosidase which
was developed specifically to differentiate V parahaemolytics from ot her bacteria by us ing a
chromogenic su bstrate instead of sugar fermentation in traditional growth med ia such as
TCBS (Hara-Kudo et 01 2001) Therefore it is more sensitiv e and accurate for identi fying V
porahllemolyticlIs in food samples rather than the use of Te ES (Oi Pinto 2010 Canizalez-
Roman 20 II)
26 Identification through Gram-stain and biochemical tests
V parahaemoiyticus is a Gram-negative bacterium th us this organi sm stained pink or red
after Gram staining (HPA 20073) Under microscopic observation a typical V
pllrahaemolyticus appeared as rod characterist ica lly curved or comma-shoped The bacterium
is also positi ve for motility under microscopic observations (ISO 2007 BPA 2007a)
V parahaemolylieus ca n be identified through a seri es of biochemical tests According
to Khan et 01 (2007) V para haemolyticus can be distinct ively differentia ted fro m the other
Vibrio spp by performing sa lt tolerance and acid fro m femlen tation tests A typical V
parahaemoiYlicus co lonies would not grow in AFW without th e presence of at least 3 of sali
10
~-------
but it can only endure the presence of salt up to 8 (ISO 2007 Khan el aI 2007) In the
other hand the fermentation test for acid production is the most important test to differentiate
between V parahaemolyliclIs and V vulnificus species (Khan el aI 2007) Jayasinge et ai
(2010) even suggested that the combination of salt toleranc e test Voges-Proskauer test and
ONPG test can provide a strai ghtforward differentiation of V parahaemoly liclis from the
other Vibrionaceae species (Figure 31)
Citrate test screens the ab ilit y of bacterial isolates to utilize ci trate as its carbon and
energy source (MacFaddin 2000) This test is a very important test to identify gram-negative
pathogens (Tang el aI 1998) The MR-VP medium which was used for Met hyl red and
Voges Proskauer test contains glucose and peptone Methyl red test screens for the bacterial
metaboli sm pathways for glucose fermentation while the Voges-Proskauer tes t is an assay for
organisms which ferment glucose with only one fermentation product usuall y acetic acid
The TS[ test is another useful test tha t can distinguish V parahaemolyticus from other
bacteria (ISO 2007) The TSJ agar med ium contains peptone fenous sulfate lactose sucrose
and a small amount of glucose (dex trose) The pH indi cator is phenol red This medium
differentiates bacteria based on their ability to fennent glucose lactose ando r sucrose and on
their abi lity to reduce sulfur to hydrogen sulfide (H S) The T SJ test also haj the abi lity to
determine the production of H]S (Kim et aI 1999)
In additi on other bi ochemica l tests such as oxidase lysine decarboxylase om ithi ne
decarboxylase ca talase and indole can also be used for the purpose of identifying V
II
-- ---- - ----------- - ~i
parahaemoyticus iso lates (ISO 2007 Jayasinghe et ai 2007 Khan et aI 2007) The
biochemical reactions o f V parahaemoiyticus are summarized in Table 21
Table 21 Siandard biochemical tesl reactions of V porahaemoyticus
TeSi S Reaction of V parahaem olyaclIs Refe rences Salt tol ance
+
Sali ne TSI agar KIA
No HS and gas production
Simmons Citrnh
Voges-Proskauer
Methyl-Red +
[SO (2007)
Jayasinge et al (2007)
Khan et of (2007)
ISO (2007)
Kim et I (1999)
Abide et til (1999)
Kim 11111 (1999)
Khan et til (2007)
Jayasinge el III (20 (0)
Kim et 0 (2 007)
Note (-) ~ nega live (+) ~ positi ve K ~ alka line ~ aC idic
12
CHAPT ER 3
MATERIALS AND METI IODS
31 Samples collection
Eighty raw vegetable samples were collected week ly from two wet markets (WI amp W2) and
two supennarkets (S 1 amp S2) in Kuching Sarawa k The collected samples consisted o f four
di fferent groups of vegetables which are regularly consumed ra w by the locals These samples
wcr~ rand omly selected within ten weeks -period which is from October 20 10 to January
20 11 Duri ng collection each sample was transferred to clean plastic bags labeled acco rding
to the place they were collected and were analyzed immed iately on arriva l to the labo rat ory
The types of raw vegetable sa mples obtained were as shown in Table 31
Tab le 31 Types o f raw vegetable samples co llected from wet markes and supcmlarkels
Group Loca l nam e English nam e Scienti fi c Home l mple coding No or samples
Soil Kubis Cabbage Brassica v eracea K 20 Roots Lobak Canmiddotot Douclfs carola C ~O
mera h Beans KaCtlO g Four-winged PsophOCaJjJ lls KB cO
bow l bean JeIlagulIolobus Shoot Tauge Bean-I_prout Vigna rad mc T 10 Total 80
13
---------~ I I
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
by fever or hypotension with the isolation of V parahaemolyticus in the blood Septicemia
typicall y occurs in person with underlying health problems such as liver disease diabetes
cancer or other immune disorder (Simjee 2007) V parahaemoyticlis can even ca use fala l
septicemia in an immune-compromised hosts
The symptoms usuall y occur on the average of 15 hours but can began as early as 4
hours and as late as 36 hours after exposure and this will continue up to 3 days (Barceloux
200S) However these illnesses are usua lly categorized as a short illnesses and no prolonged
enect will take place after recovery (Wright et al 2009)
25 Isolation of V parahael1lolyliclIs
For the isolation of V parahaemoyticus an initial emichment using alka line peptone wat er
(APW) with pH S5 would be useful as this medium supports the growth uf viable ce lls
(Simjee 2007) Plati ng of emichment broth onto TCBS agar has been widely used for
selective isolation of V parahaemolytics from foods (Thompson el ai 2006 Oi Pinto et al
201 0 Canizalez -Roman et ai 2011 ) An average of 24 hours incubation at 37 degC is requ ired
fo r typica l V parahaemolylicus to develop colonies that are smooth gree n (s u~rose nega tive)
and 2 to 3 nun in di ameter on TCBS (ISO 2007) However V parahaenlOlytlcus co lonies on
TCBS agar are difficult to distinguish vi suall y from other bacterial colonies since they can be
covered by yellow color produced by sucrose-fennenting bacteria (Thompson el 01 2006
Rambach 2007)
9
_ ------~-- i ~~
Another widely used selective medium is CHROMagar VibrioT On this medium V
pllrahaemoiy liclis will developed mau ve color colonies (Kudo et aI 2001 Oi Pi nto 2010
Canizalez-Roman 2011) This agar medium contains substrates for beta-galactosidase which
was developed specifically to differentiate V parahaemolytics from ot her bacteria by us ing a
chromogenic su bstrate instead of sugar fermentation in traditional growth med ia such as
TCBS (Hara-Kudo et 01 2001) Therefore it is more sensitiv e and accurate for identi fying V
porahllemolyticlIs in food samples rather than the use of Te ES (Oi Pinto 2010 Canizalez-
Roman 20 II)
26 Identification through Gram-stain and biochemical tests
V parahaemoiyticus is a Gram-negative bacterium th us this organi sm stained pink or red
after Gram staining (HPA 20073) Under microscopic observation a typical V
pllrahaemolyticus appeared as rod characterist ica lly curved or comma-shoped The bacterium
is also positi ve for motility under microscopic observations (ISO 2007 BPA 2007a)
V parahaemolylieus ca n be identified through a seri es of biochemical tests According
to Khan et 01 (2007) V para haemolyticus can be distinct ively differentia ted fro m the other
Vibrio spp by performing sa lt tolerance and acid fro m femlen tation tests A typical V
parahaemoiYlicus co lonies would not grow in AFW without th e presence of at least 3 of sali
10
~-------
but it can only endure the presence of salt up to 8 (ISO 2007 Khan el aI 2007) In the
other hand the fermentation test for acid production is the most important test to differentiate
between V parahaemolyliclIs and V vulnificus species (Khan el aI 2007) Jayasinge et ai
(2010) even suggested that the combination of salt toleranc e test Voges-Proskauer test and
ONPG test can provide a strai ghtforward differentiation of V parahaemoly liclis from the
other Vibrionaceae species (Figure 31)
Citrate test screens the ab ilit y of bacterial isolates to utilize ci trate as its carbon and
energy source (MacFaddin 2000) This test is a very important test to identify gram-negative
pathogens (Tang el aI 1998) The MR-VP medium which was used for Met hyl red and
Voges Proskauer test contains glucose and peptone Methyl red test screens for the bacterial
metaboli sm pathways for glucose fermentation while the Voges-Proskauer tes t is an assay for
organisms which ferment glucose with only one fermentation product usuall y acetic acid
The TS[ test is another useful test tha t can distinguish V parahaemolyticus from other
bacteria (ISO 2007) The TSJ agar med ium contains peptone fenous sulfate lactose sucrose
and a small amount of glucose (dex trose) The pH indi cator is phenol red This medium
differentiates bacteria based on their ability to fennent glucose lactose ando r sucrose and on
their abi lity to reduce sulfur to hydrogen sulfide (H S) The T SJ test also haj the abi lity to
determine the production of H]S (Kim et aI 1999)
In additi on other bi ochemica l tests such as oxidase lysine decarboxylase om ithi ne
decarboxylase ca talase and indole can also be used for the purpose of identifying V
II
-- ---- - ----------- - ~i
parahaemoyticus iso lates (ISO 2007 Jayasinghe et ai 2007 Khan et aI 2007) The
biochemical reactions o f V parahaemoiyticus are summarized in Table 21
Table 21 Siandard biochemical tesl reactions of V porahaemoyticus
TeSi S Reaction of V parahaem olyaclIs Refe rences Salt tol ance
+
Sali ne TSI agar KIA
No HS and gas production
Simmons Citrnh
Voges-Proskauer
Methyl-Red +
[SO (2007)
Jayasinge et al (2007)
Khan et of (2007)
ISO (2007)
Kim et I (1999)
Abide et til (1999)
Kim 11111 (1999)
Khan et til (2007)
Jayasinge el III (20 (0)
Kim et 0 (2 007)
Note (-) ~ nega live (+) ~ positi ve K ~ alka line ~ aC idic
12
CHAPT ER 3
MATERIALS AND METI IODS
31 Samples collection
Eighty raw vegetable samples were collected week ly from two wet markets (WI amp W2) and
two supennarkets (S 1 amp S2) in Kuching Sarawa k The collected samples consisted o f four
di fferent groups of vegetables which are regularly consumed ra w by the locals These samples
wcr~ rand omly selected within ten weeks -period which is from October 20 10 to January
20 11 Duri ng collection each sample was transferred to clean plastic bags labeled acco rding
to the place they were collected and were analyzed immed iately on arriva l to the labo rat ory
The types of raw vegetable sa mples obtained were as shown in Table 31
Tab le 31 Types o f raw vegetable samples co llected from wet markes and supcmlarkels
Group Loca l nam e English nam e Scienti fi c Home l mple coding No or samples
Soil Kubis Cabbage Brassica v eracea K 20 Roots Lobak Canmiddotot Douclfs carola C ~O
mera h Beans KaCtlO g Four-winged PsophOCaJjJ lls KB cO
bow l bean JeIlagulIolobus Shoot Tauge Bean-I_prout Vigna rad mc T 10 Total 80
13
---------~ I I
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
Another widely used selective medium is CHROMagar VibrioT On this medium V
pllrahaemoiy liclis will developed mau ve color colonies (Kudo et aI 2001 Oi Pi nto 2010
Canizalez-Roman 2011) This agar medium contains substrates for beta-galactosidase which
was developed specifically to differentiate V parahaemolytics from ot her bacteria by us ing a
chromogenic su bstrate instead of sugar fermentation in traditional growth med ia such as
TCBS (Hara-Kudo et 01 2001) Therefore it is more sensitiv e and accurate for identi fying V
porahllemolyticlIs in food samples rather than the use of Te ES (Oi Pinto 2010 Canizalez-
Roman 20 II)
26 Identification through Gram-stain and biochemical tests
V parahaemoiyticus is a Gram-negative bacterium th us this organi sm stained pink or red
after Gram staining (HPA 20073) Under microscopic observation a typical V
pllrahaemolyticus appeared as rod characterist ica lly curved or comma-shoped The bacterium
is also positi ve for motility under microscopic observations (ISO 2007 BPA 2007a)
V parahaemolylieus ca n be identified through a seri es of biochemical tests According
to Khan et 01 (2007) V para haemolyticus can be distinct ively differentia ted fro m the other
Vibrio spp by performing sa lt tolerance and acid fro m femlen tation tests A typical V
parahaemoiYlicus co lonies would not grow in AFW without th e presence of at least 3 of sali
10
~-------
but it can only endure the presence of salt up to 8 (ISO 2007 Khan el aI 2007) In the
other hand the fermentation test for acid production is the most important test to differentiate
between V parahaemolyliclIs and V vulnificus species (Khan el aI 2007) Jayasinge et ai
(2010) even suggested that the combination of salt toleranc e test Voges-Proskauer test and
ONPG test can provide a strai ghtforward differentiation of V parahaemoly liclis from the
other Vibrionaceae species (Figure 31)
Citrate test screens the ab ilit y of bacterial isolates to utilize ci trate as its carbon and
energy source (MacFaddin 2000) This test is a very important test to identify gram-negative
pathogens (Tang el aI 1998) The MR-VP medium which was used for Met hyl red and
Voges Proskauer test contains glucose and peptone Methyl red test screens for the bacterial
metaboli sm pathways for glucose fermentation while the Voges-Proskauer tes t is an assay for
organisms which ferment glucose with only one fermentation product usuall y acetic acid
The TS[ test is another useful test tha t can distinguish V parahaemolyticus from other
bacteria (ISO 2007) The TSJ agar med ium contains peptone fenous sulfate lactose sucrose
and a small amount of glucose (dex trose) The pH indi cator is phenol red This medium
differentiates bacteria based on their ability to fennent glucose lactose ando r sucrose and on
their abi lity to reduce sulfur to hydrogen sulfide (H S) The T SJ test also haj the abi lity to
determine the production of H]S (Kim et aI 1999)
In additi on other bi ochemica l tests such as oxidase lysine decarboxylase om ithi ne
decarboxylase ca talase and indole can also be used for the purpose of identifying V
II
-- ---- - ----------- - ~i
parahaemoyticus iso lates (ISO 2007 Jayasinghe et ai 2007 Khan et aI 2007) The
biochemical reactions o f V parahaemoiyticus are summarized in Table 21
Table 21 Siandard biochemical tesl reactions of V porahaemoyticus
TeSi S Reaction of V parahaem olyaclIs Refe rences Salt tol ance
+
Sali ne TSI agar KIA
No HS and gas production
Simmons Citrnh
Voges-Proskauer
Methyl-Red +
[SO (2007)
Jayasinge et al (2007)
Khan et of (2007)
ISO (2007)
Kim et I (1999)
Abide et til (1999)
Kim 11111 (1999)
Khan et til (2007)
Jayasinge el III (20 (0)
Kim et 0 (2 007)
Note (-) ~ nega live (+) ~ positi ve K ~ alka line ~ aC idic
12
CHAPT ER 3
MATERIALS AND METI IODS
31 Samples collection
Eighty raw vegetable samples were collected week ly from two wet markets (WI amp W2) and
two supennarkets (S 1 amp S2) in Kuching Sarawa k The collected samples consisted o f four
di fferent groups of vegetables which are regularly consumed ra w by the locals These samples
wcr~ rand omly selected within ten weeks -period which is from October 20 10 to January
20 11 Duri ng collection each sample was transferred to clean plastic bags labeled acco rding
to the place they were collected and were analyzed immed iately on arriva l to the labo rat ory
The types of raw vegetable sa mples obtained were as shown in Table 31
Tab le 31 Types o f raw vegetable samples co llected from wet markes and supcmlarkels
Group Loca l nam e English nam e Scienti fi c Home l mple coding No or samples
Soil Kubis Cabbage Brassica v eracea K 20 Roots Lobak Canmiddotot Douclfs carola C ~O
mera h Beans KaCtlO g Four-winged PsophOCaJjJ lls KB cO
bow l bean JeIlagulIolobus Shoot Tauge Bean-I_prout Vigna rad mc T 10 Total 80
13
---------~ I I
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
but it can only endure the presence of salt up to 8 (ISO 2007 Khan el aI 2007) In the
other hand the fermentation test for acid production is the most important test to differentiate
between V parahaemolyliclIs and V vulnificus species (Khan el aI 2007) Jayasinge et ai
(2010) even suggested that the combination of salt toleranc e test Voges-Proskauer test and
ONPG test can provide a strai ghtforward differentiation of V parahaemoly liclis from the
other Vibrionaceae species (Figure 31)
Citrate test screens the ab ilit y of bacterial isolates to utilize ci trate as its carbon and
energy source (MacFaddin 2000) This test is a very important test to identify gram-negative
pathogens (Tang el aI 1998) The MR-VP medium which was used for Met hyl red and
Voges Proskauer test contains glucose and peptone Methyl red test screens for the bacterial
metaboli sm pathways for glucose fermentation while the Voges-Proskauer tes t is an assay for
organisms which ferment glucose with only one fermentation product usuall y acetic acid
The TS[ test is another useful test tha t can distinguish V parahaemolyticus from other
bacteria (ISO 2007) The TSJ agar med ium contains peptone fenous sulfate lactose sucrose
and a small amount of glucose (dex trose) The pH indi cator is phenol red This medium
differentiates bacteria based on their ability to fennent glucose lactose ando r sucrose and on
their abi lity to reduce sulfur to hydrogen sulfide (H S) The T SJ test also haj the abi lity to
determine the production of H]S (Kim et aI 1999)
In additi on other bi ochemica l tests such as oxidase lysine decarboxylase om ithi ne
decarboxylase ca talase and indole can also be used for the purpose of identifying V
II
-- ---- - ----------- - ~i
parahaemoyticus iso lates (ISO 2007 Jayasinghe et ai 2007 Khan et aI 2007) The
biochemical reactions o f V parahaemoiyticus are summarized in Table 21
Table 21 Siandard biochemical tesl reactions of V porahaemoyticus
TeSi S Reaction of V parahaem olyaclIs Refe rences Salt tol ance
+
Sali ne TSI agar KIA
No HS and gas production
Simmons Citrnh
Voges-Proskauer
Methyl-Red +
[SO (2007)
Jayasinge et al (2007)
Khan et of (2007)
ISO (2007)
Kim et I (1999)
Abide et til (1999)
Kim 11111 (1999)
Khan et til (2007)
Jayasinge el III (20 (0)
Kim et 0 (2 007)
Note (-) ~ nega live (+) ~ positi ve K ~ alka line ~ aC idic
12
CHAPT ER 3
MATERIALS AND METI IODS
31 Samples collection
Eighty raw vegetable samples were collected week ly from two wet markets (WI amp W2) and
two supennarkets (S 1 amp S2) in Kuching Sarawa k The collected samples consisted o f four
di fferent groups of vegetables which are regularly consumed ra w by the locals These samples
wcr~ rand omly selected within ten weeks -period which is from October 20 10 to January
20 11 Duri ng collection each sample was transferred to clean plastic bags labeled acco rding
to the place they were collected and were analyzed immed iately on arriva l to the labo rat ory
The types of raw vegetable sa mples obtained were as shown in Table 31
Tab le 31 Types o f raw vegetable samples co llected from wet markes and supcmlarkels
Group Loca l nam e English nam e Scienti fi c Home l mple coding No or samples
Soil Kubis Cabbage Brassica v eracea K 20 Roots Lobak Canmiddotot Douclfs carola C ~O
mera h Beans KaCtlO g Four-winged PsophOCaJjJ lls KB cO
bow l bean JeIlagulIolobus Shoot Tauge Bean-I_prout Vigna rad mc T 10 Total 80
13
---------~ I I
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
parahaemoyticus iso lates (ISO 2007 Jayasinghe et ai 2007 Khan et aI 2007) The
biochemical reactions o f V parahaemoiyticus are summarized in Table 21
Table 21 Siandard biochemical tesl reactions of V porahaemoyticus
TeSi S Reaction of V parahaem olyaclIs Refe rences Salt tol ance
+
Sali ne TSI agar KIA
No HS and gas production
Simmons Citrnh
Voges-Proskauer
Methyl-Red +
[SO (2007)
Jayasinge et al (2007)
Khan et of (2007)
ISO (2007)
Kim et I (1999)
Abide et til (1999)
Kim 11111 (1999)
Khan et til (2007)
Jayasinge el III (20 (0)
Kim et 0 (2 007)
Note (-) ~ nega live (+) ~ positi ve K ~ alka line ~ aC idic
12
CHAPT ER 3
MATERIALS AND METI IODS
31 Samples collection
Eighty raw vegetable samples were collected week ly from two wet markets (WI amp W2) and
two supennarkets (S 1 amp S2) in Kuching Sarawa k The collected samples consisted o f four
di fferent groups of vegetables which are regularly consumed ra w by the locals These samples
wcr~ rand omly selected within ten weeks -period which is from October 20 10 to January
20 11 Duri ng collection each sample was transferred to clean plastic bags labeled acco rding
to the place they were collected and were analyzed immed iately on arriva l to the labo rat ory
The types of raw vegetable sa mples obtained were as shown in Table 31
Tab le 31 Types o f raw vegetable samples co llected from wet markes and supcmlarkels
Group Loca l nam e English nam e Scienti fi c Home l mple coding No or samples
Soil Kubis Cabbage Brassica v eracea K 20 Roots Lobak Canmiddotot Douclfs carola C ~O
mera h Beans KaCtlO g Four-winged PsophOCaJjJ lls KB cO
bow l bean JeIlagulIolobus Shoot Tauge Bean-I_prout Vigna rad mc T 10 Total 80
13
---------~ I I
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
CHAPT ER 3
MATERIALS AND METI IODS
31 Samples collection
Eighty raw vegetable samples were collected week ly from two wet markets (WI amp W2) and
two supennarkets (S 1 amp S2) in Kuching Sarawa k The collected samples consisted o f four
di fferent groups of vegetables which are regularly consumed ra w by the locals These samples
wcr~ rand omly selected within ten weeks -period which is from October 20 10 to January
20 11 Duri ng collection each sample was transferred to clean plastic bags labeled acco rding
to the place they were collected and were analyzed immed iately on arriva l to the labo rat ory
The types of raw vegetable sa mples obtained were as shown in Table 31
Tab le 31 Types o f raw vegetable samples co llected from wet markes and supcmlarkels
Group Loca l nam e English nam e Scienti fi c Home l mple coding No or samples
Soil Kubis Cabbage Brassica v eracea K 20 Roots Lobak Canmiddotot Douclfs carola C ~O
mera h Beans KaCtlO g Four-winged PsophOCaJjJ lls KB cO
bow l bean JeIlagulIolobus Shoot Tauge Bean-I_prout Vigna rad mc T 10 Total 80
13
---------~ I I
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
32 Samples preparation and enrichment
Tbe sa mple preparation methods used in thi s study was based on th e Bacteriological
AIalytical Manual standard method (Kaysncr and DePaola 2004) with slight mod ification
according to the procedures by Chai et of (2007) and Tunllng el al (20 10) A lkaline Peptone
Water (APW) was prepared ahead of samplin g by suspending 255 grams of the medium in
I L of distilled waLer with the addi tion of 3 NaCl The pH values were adjusted to 85 and
the med iu m was steril ized in autoclave at 121degC for 15 minutes As SOOn as the vegetable
samples arrived in the laboratory the sa mples were immed iately chopped into small pieces 10
faci litate crushing 25 g of eac h sample was placed in stomacher bags and homogenized in
225 ml APW The enrichment mi xtures were then incubated at 37degC for 18 to 24 hours
33 Isolation of V paraiwelll olyticlIs
The isolation of V parahaemoiylicus was achieved by using two types of so lid selecti ve
med ia namely Thios ulphate-Citrate-Bile salt Sucrose (TCBS) agar and the comm ercia lly
avai lable CHROMagar Vibrio The isolation procedu res appl ied in this tudy was based on
ISO Manua l of Standard Met hods (2007) with slight modifications
t4
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
After an ovemight incubation 10- 10- 10-4 and 10-5 dilutions were prepared
Following dilution I ~I of the dilu ted enrichment was transferred onto TCBS aga r plates and
lVas spread evenly The plates were sea led and incu bated ovemight in a 37degC incubator The
suspected V parahaemulrlicus colonies which were indicated by th e appearance o f round 2
to 3 mm in diameter and green in colo r were hather picked and streaked 0 1110 CHROMagar
VibrioTN The plates were then incubated at 37degC fo r 18 to 24 hours_
After incubation the V parahaemolyliCus iso lates on CHROMagar Vibriotrade appeared
as mauve color colonies One or more (up to three) of these typical colonies were picked and
stored in LB broth and NA slant supplemented with 3 NaCI for further usc
34 Preparation for Gram staining and biochemical tests
Before any furth er tests can be carried out the previousl y stored cuJllues were subjected for
colonies pUiflcation process Pu re cultures were obtained by sub-cu huring the stock cu lture
onto C HROMagar VibrioHl Only pure and fresh cultures were used for the subsequ ent tests _
Preparation of cultures was accompli shed by streaking a loopfu of the pure stock culture on
NA aga r (w ith 3 Nael) and the plates were incubated ovemight at 37 C Single colonies
picked from NA plales were directl y used for biochemical tests and Gran stain ing
t5
