DETERMINATION OF VIBRIO CONTAMINATION AND BACTERIAL COUNT IN

MUSSELS SOLD IN WHARFS OF SINEGUELASAN, BACOOR CITY AND AT

BERNABE, LAS PIAS CITY

An Undergraduate Thesis
Presented to the
Faculty of the College of MedicalTechnology
UNIVERSITY OF PERPETUAL HELP SYSTEM
AlabangZapote Road, Pamplona, Las Pias City

In Partial Fulfillment
Of the Requirement for the Degree
BACHELOR OF SCIENCE IN MEDICAL TECHNOLOGY

MANLAGNIT, AIRA BEATRICE A.

RACELIS, MICHELLE DIANNE A.

MARCH 2014

CHAPTER 1
THE PROBLEM AND ITS REVIEW OF RELATED LITERATURE
Introduction
According to Center for Disease Control and Prevention, Most people
dont think about food safety until they or someone they know gets sick from eating
contaminated food and it can happen to anyone.
Consumption of foods that has either been contaminated at source or which
becomes contaminated during the processing and retail chain may cause number of
bacterial illnesses. Such illnesses may ascend from infection with the bacteria
themselves or by the ingestion of toxins formed in the foodstuff prior to consumption.
Seafoods come after meat and vegetables that an individual takes in his meal.
One of the seafood typically prepared in the table are mussels. Mussels are tasty
natural food, versatile shells and can be used in many different dishes. Healthy
benefits of these are it contains vitamins, minerals and a high protein source that
boost your immune system and brain.
Mussels are filter feeders and are suited to trap all bacteria and
viruses,pathogenic or otherwise,that live in the waters, one such bacteria is Vibrio, a
genus of gram negative bacteria possessing a curved rod shape with a several
species which can cause foodborne infection. The bacteria is associated with eating
undercooked seafood and typically found in salt water. Pathogenic Vibrios in humans
include V. cholerae (causative agent of cholera), V. parahaemolyticus, and V.
vulnificus. All of them have an absolute requirement for Na + for growth although
some, such as V. cholerae only require trace amounts. The surface and intestinal

microbial flora of harvested fish and shellfish, particularly those from brackish,
estuarine and coastal environments, will tend to contain high numbers of members of
the genus Vibrio. Only a small proportion of the vibrio present will belong to species
that may be pathogenic in humans and, of these, only a small proportion of the
isolates of such species may possess the pathogenicity traits that enable them to
colonize and cause disease in the human body.

This study will attempt to determine Vibrio contamination on the mussels that
are harvested and sold in wharfs of Sineguelasan, Bacoor City and Bernabe, Las
Pias City.
Review of Related Literature and Related Studies
Wharf Profiles
Bacoor is a first class urban component city in the province of Cavite. It is a
well-known place for mussels and oysters. The seafood wharfs are located mostly in
West Bacoor District 1 specifically Sineguelasan, Alima and Digman. Sineguelasan
is the most popular seafood stall of several mussels and oysters while Alima and
Digman are both private seaports. The three seaport areas have the specific time in
collecting and harvesting the mussels and oysters which is around 5:00 am, while
the time of arrival in the market is around 2:00-4:00pm. The primary delivery site of
harvested mussels and oysters are in the cities of Malabon and Navotas.
Las Pias City is one of the cities in the National Capital Region of the
Philippines. It has a fishport located in Bernabe Compound, Pulang Lupa I that arose
from a corollary project of Las Pias government. This is where fishermen dock their

bancas with seafoods like fishes and mussels then deliver and sell it on nearby stalls
and in Zapote, Las Pias public market.
Mussels
Mussels are molluscs and are related to water snails. Because their shells are
divided into two halves, they are called bivalves. The shell protects the soft body of
the animal. A powerful foot enables the animal to change its position. Mussels suck
in water and extract the oxygen and food that they require. There are both marine
and fresh water mussels and can be found worldwide. The marine mussels prefer
cool seas. Freshwater mussels, also known as naiads, inhabit streams, lakes and
ponds and there are around 1,000 known species. The shells are dark blue or dark
greenish brown on the outside, while on the inside they have a pearly appearance.
Mussels attach themselves to solid objects or to one another by strands called
byssus threads and often appear in dense clusters.
Mussels as filter feeders, filters bacteria, plankton and detritus from the water,
when large beds of this gregarious species form, individuals are bonded together
with threads of byssus. Organisms that attach to mussels, such as seaweeds and
barnacles may increase the risk of the mussel becoming detached by wave action,
however mussels are able to sweep their foot over their shell which may help
minimize likehood of such organism becoming attached. The sexes are separate,
fertilization occurs externally with spawning peaks in spring and summer. The larval
stage is free-swimming and planktonic for around 4 weeks before settling first on
filamentous organisms such as seaweeds. After growing for a while, they detach and
drift in the water on a long byssal thread; a mode of disposal likened to that of young
spiders floating through the air on a silk thread. After four weeks or so, young mussel

will have settled again, this time on a mussel bed. Young mussels are thought to
have evolved primary settlement on filament substrates in order to avoid having to
compete with adult mussels. (eol,2010)
Vibrios
Vibrio spp. is oxidase-positive, glucose fermenting, gram negative bacilli that
grow on MacConkey agar. Brackish or marine water is their primary habitat.
Transmission to humans is by ingestion of contaminated water or seafood or by
exposure of disrupted skin and mucosal surfaces to contaminated water.
(Forbes,2007)
Vibrio species are curved; Gram negative rods that produce colonies 2-3 mm
in diameter on blood agar and colonies on thiosulphate citrate bile salt sucrose
(TCBS) are either yellow or green. Vibrio species are facultative anaerobes, motile
by a single polar flagellum, and are oxidase-positive (except Vibrio metschnikovii).
They are usually sensitive to the vibrio static agent O129 (2, 4-diamino-6, 7diisopropylpteridine phosphate-150 g disc). Growth is stimulated by sodium ions
(halophilic) - the concentration required is reflected in the salinity of their natural
environment. V. cholerae (the causative agent of cholera) is not halophilic. Doubling
time of vibrios under ideal conditions may be as short as 10 minutes or less, but
usually much longer in seafoods at ambient temperature. Little growth of pathogenic
vibrios usually occurs in seafood stored below 10C. A one-log reduction of
pathogenic vibrios in seafoods takes between 1 and 10 minutes at 50C, depending
on species and strain. Vibrios die out gradually at refrigeration temperatures and
numbers are markedly reduced by freezing and thawing. (Borresen,2008)
Vibrio spp.

Vibrio cholerae
V. cholerae also called Kommabacillus is a gram negative, facultative,
comma shaped bacterium with a polar flagellum. Growth of V. cholerae is stimulated
by addition of 1% sodium chloride (NaCl). However, an important distinction from
other Vibrio spp is the ability of V. cholerae to grow in nutrient broth without added
NaCl. Optimal temperature for growth is 37C, with growth possible between 1043C. The optimal pH is 7.6, growth possible at pH 5.0-9.6. It yields large yellow
colonies in TCBS appear

medium to large ,smooth, opaque, iridescent with a

greenish hue. It strains have two types: serogroups and non-serogroups of V.

cholera O1 and O139.The most wide sweeping pathogenic strain is the Vibrio
cholerae serotype O1 El Tor N16961 strain that causes the pandemic disease
cholera. The Non-O1 and non-O139 V. cholerae can cause mild diarrhea but do not
generate epidemics. The bacteria infect the intestine and increases mucous
production causing diarrhea and vomiting which result in extreme dehydration and, if
not treated will cause death. It is usually transmitted through the feces of an infected
person, and sometimes by way of unclean drinking water or contaminated food.
Vibrio parahaemolyticus
V. parahaemolyticus is a Gram-negative, straight or curved motile rod. It is
halophile (requiring 1 to 3 percent NaCl) and will grow in 7.0 percent NaCl. Optimal
temperature for growth is 35 to 37C, but it will grow over a range off 22 to 42C.
Growth is inhibited below pH 5.0 and above pH 11.0. V. parahaemolyticus does not
grow well on some of the differential media used to grow Salmonella and Shigella,
but it does grow well on blood agar. It also grows well on TCBS, where it yields
green colonies. V. parahaemolyticus is usually identified by its oxidase- positive

growth on blood agar. It lives in brackish saltwater and causes gastrointestinal illness
in humans. Outbreaks of V. parahaemolyticus gastroenteritis have received
considerable attention in Japan, where it is one of the most commonly occurring
food-poisoning syndromes.The seafood bivalve molluscs, finfish and crustaceans
which may be contaminated of V. parahaemolyticus and served raw or superficially
cooked are exposing to bacterial diseases.

Vibrio vulnificus
V. vulnificus can cause severe wound infections, bacteremia and probably
gastroenteritis. It is free-living estuarine bacterium. Infections have been reported
from Korea and the organisms may be distributed worldwide. V. vulnificus is
particularly apt to be found in oysters, especially in warm months. Bacteremia with
no focus of infection occurs in persons who have eaten infected oysters and who
have alcoholism or live disease.

Wounds may become infected in normal or

immunocompromised persons who are in contact with water where the bacterium is
present. About 50% of the patients with bacteremia die because the infection
precedes rapidly.This virulent, gram-negative bacterium causes two distinct
syndromes. The first is primary septicemia caused by consuming raw or
undercooked seafood, particularly raw oysters. The second is a necrotizing wound
infection acquired when an open wound is exposed to warm seawater with high
concentrations of V. vulnificus. In diagnosis, culturing of organisms on standard
laboratory media, thiosulphate citrate bile salt sucrose (TCBS) is preferred medium

for stool cultures, where most strains produce blue-green (sucrose-negative)

colonies.
Vibrio mimicus
V. mimicus is a bacterium that resembles V. cholerae in many characteristics,
including its ability to grow in low concentrations of sodium and its antigenic
structure. It has been implicated in individual cases and outbreaks of gastroenteritis
and some strains can also produce cholera toxin. V. mimicus produce colonies of 2 3 mm in diameter on blood agar, and colonies on (TCBS) are of green color.
Vibrio fluvialis
V. fluvialis is an inhabitant of estuarine and coastal waters and has been
identified as causing individual cases and outbreaks of gastroenteritis. As with many
other vibrios, infection is usually associated with the consumption of raw or
undercooked seafood, especially raw oysters.
Vibrio furnissii
V. furnissii is closely related to Vibrio fluvialis both genetically and
phenotypically. However, it has been isolated much more rarely from fecal samples
of patients with gastroenteritis and so there is some doubt as to its significance as a
seafood-associated pathogen.
Vibrio hollisae
V. hollisae is a marine vibrio that has been reported to cause gastro-enteritis
following consumption of raw shellfish. The significance of this vibrio is difficult to
estimate because of its failure to grow on conventional vibrio media. In general,

contamination at source is a greater problem with respect to bivalve molluscs than

other seafood due to their filter-feeding activities which tend to concentrate
pathogens within the gut of the bivalves. Following harvest, these tend to be eaten
raw or lightly cooked, thus potentially allowing the survival of any pathogens present
in the bivalves at harvest.
Since Vibrios are all resistant to salt they are not inhibited by salt
preservations. Symptoms appear two to 48 hours after ingesting the contaminated
food. They include: gastroenteritis, vomiting, diarrhea, fever and chills. Foods
implicated are: raw or improperly cooked fish and seafood.
Laboratory tests
Most Vibrio spp. are oxidase and nitrate positive (V.metschnikovii is oxidase
and nitrate negative).Immunodiagnostic and molecular diagnostic procedures are
available for diagnosis of cholera.Commercial products are available for diagnosis of
cholera toxin.Once isolated.Vibrio spp are identified using commercial identification
systems.Antisera are available for i9dentification of V.cholerae O1 and O139. The
string test can be used to differentiate between Vibrio cholera (string positive test)
and other Vibrio spp(string test negative).To perform the string test, colonies from an
18-24 hour culture of the isolate are mixed into a drop of 0.5% sodium deoxycholate,
and DNA is released from the cells, causing the mixture to become viscious. The tip
of a wire inoculating loop is immersed into the mixture and slowly drawn away.In a
positive test, a long mucoid string will cling to the wire loop.

Related Studies
Popovic and co-workers (2010) randomly selected and collected 240
samples of fresh and frozen seafood from market in Croatia on warm and cold
seasons of the year, analyzed it for microbiological quality. Samples were
homogenized in 225 ml of 3% NaCl containing alkali peptone solution and
were incubated for 6 h at 37C for frozen seafood and at 22C for fresh
seafood. Afterwards of incubation, 1 ml of alkaline saline peptone water was
placed in a tube containing 10 ml of alkaline saline peptone water for 18 h at
41.5C before being inoculated onto TCBS for 24 h at 37C facilitating
identification of suspect Vibrio isolates. Microbiological analysis results
showned 5% overall prevalence of V. parahaemolyticus,V cholera and V.
alginolyticus were not presented here. Fresh and frozen shellfish in both
seasons recovery rate was high except for fresh fish sample in summer.
Therefore V. parahaemolyticus was more pronounced in fresh species without
regard to the season.

Tayo and his team (2011) determined the incidence of potentially pathogenic
Vibrio Spp. in fresh seafood from Itu Creek in Uyo, Akwa Ibom State, Nigeria.
Samples of sea foods analyzed microbiologically in this study showed varying
degree of Vibrio contamination. The study showed that skin, gills and intestine of fish
used in this study were heavily loaded with different species of Vibrio. This study
showed that a total of 79 isolates of Vibrio spp. were obtained from 150 samples of
fresh seafoods with Vibrio cholera as the most predominant. This was followed by
Vibrio mimicus, V. parahaemolyticus, Vibrio fluvialis. It showed that Vibrio vulnificus
was least predominant. This is comparable with the findings of some other authors in
a similar study. Yang and co-workers (2008) revealed 251 isolates of V.
parahaemolyticus from 1293 seafood samples (19.0%) collected from the 25 sites in
China, during July to October in 2007, while Jiet and others ( 2011) reported 58.6%
of 239 samples from different sources were positive for V. vulnificus in 10 Chinese
cities from June to September 2009. Compared to other studies, nearly similar
results were detected by a study in Turkey determining the presence of Vibrios in
66.0% of samples and in Alexandria, North Coast of Egypt Mediterranean Seas
determining the presence of Vibrios in 52.0% of samples. In a study by Merwad and
co-associates (2011) the overall prevalence of zoonotic vibrios was 57.3% in white
shrimps, 48% in blue crabs and 54% in oysters. Pinto and co-workers (2008)
reported 32.6% for mussels in Italy; Blanco Abad and others (2009) reported
11.2% for mussels in Spain and Kirs and co-associates ( 2010) reported 94.8% for
oysters in New Zealand.
Vibrios are associated with live seafood as they form part of the indigenous
microflora of the marine environment. Foodborne infections with Vibrio spp. are

common in Asia. In Hong Kong, V. parahaemolyticus continued to be the top

causative agent among all the reported food poisoning outbreaks in recent years.
According to the figures provided by the Department of Health (DH), confirmed V.
parahaemolyticus food poisoning outbreaks affecting 2725 persons were reported
during 1999 to 2003. Among these 552 outbreaks, 313 (56.7%) were due to
consumption of seafood. Inadequate cooking (59.7%) and contamination by raw food
(23.6%) were the main contribution factors for these cases. Cholera appears as
sporadic diseases in Hong Kong. Information from the DH showed that there were
totally 49 local cholera cases reported during 1999 to 2003. The suspected food item
was identified in about half of the cases, of which, seafood accounted for about 80%.
Department of Health, HKSAR Government (2008) took water samples from typhoon
shelters and shoreline waters and found higher chance of detecting V. cholerae from
open waters and fish culture zones. Water samples which were tested positive for V.
cholerae have higher E. coli counts. It then implied that if V. cholerae is the concern
for the abstraction of seawater for keeping live seafood, both the site where the
water is abstracted and the E. coli count are important parameters to be considered.

Research Paradigm
(Independent Variable)

Collection of mussel samples

sold in wharfs of Sineguelasan,
Bacoor City and Bernabe, Las,
Pias City

(Dependent Variable)

Isolation and Bacterial count

Theoretical Framework
According to Trtanj , (2010) Vibrio strains are the most common cause of
seafood borne disease and death. It is associated with 95% fatalities from seafoodborne illnesses and wound infections.
Nambabi and his co-authors (2006) stated that vibrios played significant role
in human history. Outbreaks of cholera, caused by Vibrio cholerae, can be traced
back in time to early recorded descriptions of enteric infections. Vibrios have also
received the attention of marine microbiologists who observed that the readily
cultured bacterial population in near-shore waters and those associated with fish and
shell fish were predominantly Vibrio species. Vibrio species are mainly responsible
for causing cholera and food poisoning in humans. Vibrio cholera causes cholera
due to the intake of contaminated food such as raw oysters. Vibrio parahaemolyticus
is a major cause of food borne infections, causing food poisoning. Since Vibrio
species naturally occur in sea water, worth special mention is their need for sodium
chloride, although some species can grow with minimum sodium chloride
concentration.
Based on the study of Amira and co-associates (2013) presence of other
pathogenic bacteria isolated on TCBS media in sea food and sea water should be
considered as a signal of danger for consumer and manipulators; it suggests the

need to establish measures and requirements in order to prevent all risks related to
these sea products by biological contaminants eating those belonging to Vibrio
species.

Statement of the Problem

The study will be conducted in order to determine Vibrio spp. contamination
and bacterial count in mussels sold in wharves of Sineguelasan, Bacoor City and
Bernabe, Las Pias City .
Specifically, this study will attempt to:
1.)

Determine the presence of Vibrio spp. on mussels from both wharves.

2.)

Identify the Vibrio spp. isolated from mussels.

3.)

Determine bacterial count of Vibrio spp.isolated from mussels.

Hypothesis:
There is no significant difference between vibrio contamination and bacterial
count of mussels sold in the wharves of Sineguelasan, Bacoor,City and Bernabe,
Las Pias City.
Significance of the Study
The research conducted to the benefits of the following:

Mussel vendors. They would be conscious of aspects that could possibly

affect their products, so that they can lessen exposure of their consumers to
diseases carried by their products.
Mussel buyers and consumers. They will be able to distinguish whether
which wharf sells their mussels safer for human consumption, and help them prevent
acquiring foodborne diseases also making them more responsible in handling and
preparing foods before eating or consuming it.
Scope and Limitation
The motivation of the study is to determine the vibrio and its bacterial
count consumed by the mussels. The study will be lasted from June to September. A
total of 120 mussel samples will be collected from Sineguelasan, Bacoor and
Bernabe, Las Pias City wharfs for laboratory analysis. Homogenization in each
mussel will be the technique for isolating the specimen and for bacterial growth will
be thiosulfate citrate bile salt sucrose agar (TCBS). After the vibrio species are
identified, the MPN (Most Prabable Number) bacterial count will determined which
vibrios are more predominant.

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