Microbial Pathogenesis 99 (2016) 5e13

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Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath

Isolation and identification of Vibrio cholerae and Vibrio

parahaemolyticus from prawn (Penaeus monodon) seafood:
Preservation strategies
P.R. Yaashikaa a, A. Saravanan b, P. Senthil Kumar b, *
a
Department of Biotechnology, Bannari Amman Institute of Technology, Sathyamangalam, 638401, India
b
Department of Chemical Engineering, SSN College of Engineering, Chennai, 603110, India

a r t i c l e i n f o a b s t r a c t

Article history: Bacterial diseases are one of the major problems which affects the production, development and
Received 18 June 2016 expansion of aqua culture. Vibrio sp. are widespread in marine and estuarine environments. The several
Received in revised form pathogenic species are commonly associated with outbreaks of Vibrio species and it is mainly associated
20 July 2016
with food poisonings. In this research, the occurrence of Vibrio sp. was studied by the isolation and it is
Accepted 21 July 2016
Available online 22 July 2016
confirmed by the biochemical methods. The growth rate was studied by changing the different operating
parameters. Isolation studies were done by using enrichment and selective plating methods. The
different biochemical test was carried out and inferred that the isolated organisms were Vibrio choleraee
Keywords:
Seafood
and Vibrio parahaemolyticus. The antibiotic study was also performed to find out the resistant and
Vibrio sp sensitivity of the Vibrio species. From the results, it was observed that this can be able to correlate the
Biochemical growth of vibrio species to a limited condition and other environmental parameters for which it will be
Antibody able to find the remedial measures to prevent the growth and spreading of the diseases. Also the
Preservation different preservation method was carried out to suppress the growth rate of Vibrio sp.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction concentration. The occurrence of Vibrio sp in raw sea food is com-

Seafood and fish was found to be an important food component
for a large section of world population. Sea foods are prone to
bacterial contamination, pathogens may be present at low levels
when fish or shell fish are harvested, and others may be introduced
during handling and processing or by unsanitary practices. Such
risk is further increased if the food is mishandled during processing
where pathogens could multiply exponentially under favorable
conditions [1,5,9,24]. The World Health Organization (WHO) de-
fines food borne illness as a disease which is caused through the
consumption of contaminated food [23]. Seafood especially shells
fish is a food substrate for some zoonotic Vibrio sp. These micro-
organism causes substantial number of food borne illness
[4,17e19]. Vibrio sp are Gram-negative, facultative anaerobic
motile, curved rods with a single polar flagellum [2,3,6]. Vibrio sp
are ubiquitous bacteria and abundantly present in aquatic envi-
ronments. These bacteria are particularly resistant to high salt
* Corresponding author.
E-mail address: psk8582@gmail.com (P.S. Kumar).
mon, especially seafood from region with temperate climates
around the world from both natural and farm environments and all
sea food types. However, most surveys are qualitative which causes
difficulties in evaluating the risk relating to Vibrio sp in raw sea
food. Different species within the genus Vibrio are associated with
food borne infections and food spoilage. Among the members of
the genus, twelve species are recognized as human pathogens such
as Vibrio Choleraee, Vibrio carchariae, Vibrio mimicus, Vibrio vulnifi-
cus, Vibrio metschnikovii, Vibrio parahaemolyticus, Vibrio cincinna-
tiensis, Vibrio alginolyticus, Vibrio hollisae, Vibrio furnissii, Vibrio
damsel, Vibrio fluvialis ([7,8]). Amognst that, Vibrio Cholerae and
V. Parahaemolyticus are opportunistic pathogens and responsible
for the most cases of food borne illness. V. cholerae and
V. parahaemolyticus are recognized as serious human pathogens.
V. cholerae is a motile, rod shaped and gram negative bacteria. These
bacteria exist as natural inhabitants of aquatic ecosystems, thus
making them facultative human pathogens [14,22]. V. cholerae is
transmitted through ingestion of food or water contaminated with
the bacterium, especially via feces or vomits of infected persons,
directly or indirectly [10,16]. V. cholerae is a mesophilic organism
that grows in the temperature range of 10e43
C, with optimum

http://dx.doi.org/10.1016/j.micpath.2016.07.014
0882-4010/© 2016 Elsevier Ltd. All rights reserved.
6 P.R. Yaashikaa et al. / Microbial Pathogenesis 99 (2016) 5e13
growth at 37
C. The pH optimum for growth is 7.6 although it can
grow in the pH range of 5.0e9.6. V. cholerae can grow in the salt
range of 0.1e4.0% NaCl, while optimum is 0.5% NaCl.
V. parahaemolyticus is a rod shaped, slightly halophilic, gram
negative and non-spore forming bacterium. V. parahaemolyticus is
broadly distributed in marine environment and has been recog-
nized as a major cause of food borne illness such as diarrhea and
gastroenteritis resulting from the consumption of raw, under-
cooked or contaminated sea foods. The growth of Vibrio sp is
majorly depending on temperature of water. The Growth of path-
ogenic vibrios occurs optimally at around 37
C although the
maximum and minimum growth temperatures are 43
C and 5
C
respectively. The illness caused by V. parahaemolyticus food
poisoning is gastroenteritis characterized by watery diarrhea and
abdominal cramps in most cases, with nausea, vomiting, fever, and
headache [11e13].
The aim of this present study was isolation of Vibrio sp which
was present in the prawn seafood (Penaeus monodon) and some
preservation techniques have been employed to reduce the growth
of Vibrio. Sp such as drying, radiation, organic acid and osmotic
pressure. There is a limited range of techniques currently employed
to preserve food. The drying of fish is a well-understood physical
process. Microbial spoilage can be reduced by drying. Radiation is
effective for ensuring the microbiological safety of food naturally
contaminated by Vibrio sp [15]. NaCl stimulates the growth of all
species and is an obligate requirement for some.
2. Materials and methods
2.1. Study area and sample collection
The Vibrio sp were isolated from the prawn (Penaeus monodon).
Prawn samples were collected from kovalam beach market, Chen-
nai. It has been transported immediately within 2 h to the Envi-
ronmental laboratory, SSN College of Engineering for
bacteriological investigation. The collected samples were subjected
to qualitative and quantitative analysis for Vibrio species.
2.2. Quantitative analysis
2.2.1. Enrichment method
Test samples were weighed (25 g) and transferred aseptically to
a presterilized conical flask containing 225 mL of the alkaline
peptone water with 3% NaCl and kept in the temperature controlled
horizontal bench shaking incubator (Orbitek, India) for about
10e15 min. After 24 h duration 1 mL of inoculum was transferred to
a sterile petridish and plated by using the selective media such as
TCBS (Thiosulfate Citrate Bile Salt Sucrose Agar). The plates were
incubated at 37
C for 24 h. Isolated colonies with differences in
morphological features were transferred into nutrient agar slants.
2.3. Staining method
2.3.1. Gram staining
Gram staining is a differential staining technique employed for
studies of bacterial morphology. The smear was fixed on a clean
glass slide and it was covered by using crystal violet stain for about
30e60 s. After the time interval, the slide was cleaned by placing
under the tap water. Then, Grams iodine mordent was poured on
slide and kept for 30e60 s. Then, the smear was decolorized by
using ethanol and washed under tap water. The smear was covered
with counter stain safranin for about 60 s. After the time interval,
the slide was cleaned by placing under the tap water. Finally, the
slide was air dried and smear was placed in the microscope under
oil immersion.
2.4. Motility (hanging drop method)
Motility of the bacteria was checked by “hanging drop method”.
A loop full of 24 h culture was placed on the center of the cover slip.
A drop of Vaseline is placed on the four corners of cover slip. A
cavity slide was kept over the drop in such a way that the drop
comes with in the cavity. The Vaseline makes the cover slip to
adhere to the slide. Then the whole preparation was quickly
inverted so that the drop of culture was seen hanging from the
cover slip.
2.5. Biochemical characterization
2.5.1. Metabolic activities
(i) Starch Hydrolysis
Starch agar medium was prepared, sterilized and poured in to
petri plates. The test culture was streaked on the medium and
incubated at 37
C for 24 h. Then place 2 or 3 iodine crystals in the
Petri dish cover. After inverting the plates, iodine gets vaporized.
The clear zone outside the area of bacterial growth indicates extend
of starch hydrolysis.
(ii) Casein Hydrolysis
Casein agar medium was prepared, sterilized and poured into
petri plates. The culture was incubated at 37
C at 24 h. The test
culture was streaked on the medium and incubated. Due to the
production of casienase enzyme by bacterium, medium surround-
ing the colony become clear since the exoenzymes has converted
larger protein molecules into smaller molecules i.e. amino acid,
which makes then invisible.
(iii) Gelatin Hydrolysis
The gelatin medium prepared and sterilized by tyndallization,
keeping the medium in steaming for 3 consecutive days. Then the
medium was inoculated heavily with the test organism by the stab
inoculation and incubated for 96 h. After the incubation period the
hydrolysis of the gelatin was indicated by the liquefaction of
gelatin. The culture was incubated at room temperature and chilled
until the control solidifies before observations of liquefactions were
made.
2.6. Fermentation reactions
2.6.1. Sugar fermentation (carbohydrate fermentation test)
Carbohydrate acts as the main source of energy. The media was
prepared and poured in to test tubes, introducing Durham's tube in
to each tube without air bubbles. The tubes containing the media
were sterilized by autoclaving at 15 lbs pressure. The culture was
inoculated in to the tube and incubated in for 24 h.
2.6.2. Oxidation fermentation test (Hugh and Leifson's test)
This experiment was done to confirm the ability of the isolates
to ferment glucose with the production of acid and gas. This
experiment was carried out in a medium with phenol red indicator.
The medium was prepared and poured in to sterilized test tubes.
After cooling the medium culture was inoculated into it and inoc-
ulated. After incubation the change in color was noted. The positive
reaction was show by the color change from red to yellow.
2.6.3. Indole production
The testing for indole production is important to know the
 P.R. Yaashikaa et al. / Microbial Pathogenesis 99 (2016) 5e13
tryptophanase production. The test organism was grown in tryp-
tone media. Indole is a component of the amino acid tryptophan.
Kovac's reagent which contains the Para di methyl amino benzal-
dehyde, detects indole production. This reacts with indole to pro-
duce a red color compound. The test tubes containing tryptone
were inoculated with the inoculums and incubated at 37
C for 2e4
days and indole production was tested by adding 2 to 3 drops of
Kovac's reagent. The appearance of cherry red color indicates the
presence of indole.
2.6.4. Methyl red test
The methyl red test determines the ability of organism to acidify
a phosphate buffered glucose-peptone medium to pH 4.4 or below
and culture was inoculated and incubated at room temperature for
48 h. After incubation, two drops of methyl red solution, prepared
by dissolving 0.1 g of methyl red in 300 mL ethyl alcohol and
200 mL distilled water, was added to the inoculated medium. A red
color indicates positive reaction while yellow color signifies nega-
tive reaction.
2.6.5. Voges-Proskauer test
MR-VP broth was prepared and sterilized and then incubated at
room temperature for 24e48 h. Baritt's reagent (0.6 mL of 5% so-
lution of naphthol in ethanol and 0.2 mL of 40% potassium Hy-
droxide) was added to the cultured broth it is based on the principle
that under alkaline conditions and exposure to air, the acetoin
produced from the fermentation glucose is oxidized to diacetyl,
which forms a pink colored compound with 40% KOH. Develop-
ment of pink color within 5 min indicates a positive result.
2.6.6. Citrate utilization
Simmon's citrate agar medium was prepared and sterilized at 15
lbs for 15 min and poured as slants. These slants were streaked with
the culture and were incubated at 37
C for 24 h. The citrate test is
used to determine the ability of a bacteria to utilize a soul source of
carbon. Bacteria can break the conjugate base the salt of citrate into
organic acids and carbon dioxide. Carbon dioxide can combine with
sodium from conjugate basic salt to form basic compound sodium
carbonate a pH indicator. Bromothymol blue in the medium detect
presence of this compound by turning blue.
2.7. Respiratory reaction
2.7.1. Catalase test
During aerobic respiration in the presence of oxygen, microor-
ganisms produce hydrogen peroxide (H2O2) which is lethal to the
cell. The enzyme catalase present in some microorganisms breaks
down hydrogen peroxide to water and oxygen and helps them in
their survival. Catalase test is performed by adding H2O2 to the
culture. Release of oxygen gas (O2[) bubbles is a positive catalase
test. The fresh clean glass slide was taken and a drop of H2O2 was
placed at the center on glass slide. A loop full of Vibrio sp culture
was placed on the H2O2 drop and the appearance of bubbles was
noticed.
2.7.2. Oxidase test
A piece of filter paper was placed in a clean Petri dish and 2 or 3
drops of freshly prepared oxidase reagent was added. The colony of
indole-positive bacteria was smeared on filter paper.
2.7.3. Urease test
The urea agar medium was prepared and sterilized. The sterile
medium was poured into the tubes and slants were prepared using
aseptic technique, the culture was inoculated on to the slant. The
tubes were kept for temperature controlled horizontal bench
7
shaking incubator (Orbitek, India) for 24e48 h at 37
C. After in-
cubation, the tubes were observed for the presence of urease. The
positive reaction was shown by pink coloration.
2.7.4. Nitrate reduction test
The ability to reduce nitrate was tested in an ordinary nitrate
medium with peptone broth containing 0.3% potassium nitrate. The
prepared broth was inoculated with loop full of inoculums.
Turbidity was checked after the incubation period and nitrate
reduction was done with nitrate reagent. Alpha napthyl amine
(Reagent A) sulphanilic acid (Reagent B) was mixed in equal pro-
portion. After mixing, red color obtained was recorded indicating
the reduction of nitrate to nitrite.
2.8. Triple sugar iron agar test (TSI)
In this test a 100 mL of triple iron agar media was prepared,
dispersed in to different at 15 lbs for 15 min. After sterilization
slants were made. The slants were inoculated with test cultures by
means of stab method the tubes were incubated for 2 h at 37
C,
after the incubation period, the color of both slant and butt were
observed. H2S production was indicated by the presence of black
color. Gas production was indicated by the presence of raised butt.
Acid production was indicated by the color change of the slant and
butt.
2.9. Amino acid utilization test
Decarboxylase broth was prepared and bromocresol purple was
added as indicator. Adjusted pH and the required amino acid such
as L-Methonine Hydrochloride & L-Arginine Hydrochloride (0.5%)
were added, dissolved and distributed in small test tubes, sterilize
at 115
C for 20 min each tube was labelled to identify the different
amino acids taken. The broth was then inoculated with the cultures
and one test tube was kept as control. The reaction was carried out
in anaerobic condition by closing the surface of the medium in the
test tube with sterile liquid paraffin after inoculation.
2.10. Antibiotic sensitivity test
The test culture was transferred into a sterilized broth. The broth
is then incubated for few h at 35
C till it becomes slightly turbid. By
using a sterile cotton swab the standardized bacterial test sus-
pension was inoculated evenly on the entire surface of sterile
Muller Hinton Agar plates. Antibiotic discs were placed on the
surface of the medium and plates were incubated on 37
C for 24 h.
The antimicrobial activity was interpreted from the diameter of
zone of inhibition which was measured in millimeter.
2.11. Factors affecting growth and survival of vibrio in seafood
2.11.1. Effect of temperature
The nutrient broth was taken in a boiling tube and sterilized. The
organism was inoculated in the medium at different temperatures
(0
C, 37
C, 50
C and 70
C) and incubates. Growth of organisms
was observed at 620 nm at regular intervals of time.
2.11.2. Effect of pH
The nutrient broth was taken in a boiling tube and sterilized. The
organism was inoculated at different pH (3, 5, 7, and 9) and incu-
bated at 37
C for 24 h. Growth of organisms was observed at
620 nm at regular intervals of time.
2.11.3. Effect of salinity
The nutrient broth was taken in the boiling tube at different
8 P.R. Yaashikaa et al. / Microbial Pathogenesis 99 (2016) 5e13
concentration of NaCl (0.5%, 1%, 2% and 3%). The organism was
inoculated in the medium and incubated. Growth of organisms was
observed at 620 nm at regular intervals of time.
2.12. Different preservation method
In the present study, the four types of preservation method was
carried out and these are as follows:
2.12.1. Organic acid method
(i) Acetic acid
10 mL of acetic acid was taken in a boiling tube and the prawn
was dipped into it and kept for incubation. The prawn was grinded
in alkaline peptone water and incubated for 24 h at 37
C. Streak
plate was carried out and the result was observed.
(ii) Oleic Acid
10 mL of oleic acid was taken in a boiling tube and dipped the
prawn into it and kept for incubation. The prawn was grinded with
alkaline peptone water and incubated for 24 h at 37
C. Streak plate
was carried out and the result was observed.
(iii) Oxalic acid
10 mL of oxalic acid was taken in a boiling tube and dipped the
prawn into it and kept for incubation. The prawn was grinded in
alkaline peptone water and incubated for 24 h at 37
C. Streak plate
was carried out and the result was observed.
2.12.2. Osmotic pressure (NaCl)
Different concentration of NaCl was taken in different boiling
tube and dipped the prawn and incubated. The prawn was grinded
in alkaline peptone water and incubated for 24 h at 37
C.
Streak plate was carried out and the result was observed.
2.12.3. Radiation
The prawn was taken and kept it under UV radiation. The prawn
was grinded in alkaline peptone water and incubated for 37
C at
24 h. Streak plate was carried out and the result was observed.
2.12.4. Drying
The prawn was taken and kept for drying. The prawn was
grinded in alkaline peptone water and incubated for 37
C at 24 h.
Streak plate was carried out and the result was observed.
3. Results
3.1. Quantitative analysis
The Vibrio species obtained in the present study were isolated
from the Prawn. The samples were collected from kovalam beach
market, chennai. The quantitative analysis (presence absence test)
is used to identify the isolates.
The Total Plate Count (TPC) was retrieved using TCBS agar
following the standard procedure. The counts of various samples
were estimated using the formula.
Count  Dilution
TPC=gm ¼
Volume of water=Weight of sample
Fig. 1 shows the occurrence of Vibrio species in the TCBS agar and
the quantitative distribution of Vibrio in the prawn sample were
obtained and the result was shown in Table 1.
3.2. Qualitative analysis
The morphological characters of Vibrio sp such as size, shape
and elevation on TCBS plates were observed and the result was
shown in Table 2.
3.3. Gram staining
By gram staining procedure, all the isolates were found to be
gram negative short curved rods. Further confirmation of Vibrio sp
was made according to the biochemical analysis.
3.4. Motility test
Motility of the bacteria was checked by hanging drop method
and was found to have a darting type movement.
3.5. Biochemical characterization
3.5.1. Metabolic activity
(i) Starch hydrolysis
All the 2 isolates were tested for the starch hydrolysis and the
result was shown in Fig. 2. Both the isolate showed a clear zone
around the line streak, when the iodine was added, which indicate
a positive result.
(ii) Gelatin hydrolysis
Vibrio strains were found negative reaction for gelatin hydro-
lysis, which was indicated by the semi solid medium.
(iii) Casein hydrolysis
Casein hydrolysis was performed for all the isolates, no zone
formation around the line of streak indicating a negative result by
all the isolates.
Table 3 shows the results of biochemical characterization of
Vibrio sp.
3.6. Fermentation reaction
3.6.1. Sugar fermentation (carbohydrate fermentation)
The isolates were tested for sugar fermentation with four sugars
such as lactose, sucrose, maltose and dextrose. All the isolates were
found to be positive for sucrose, maltose lactose and dextrose and
the results were shown in Fig. 3a.
3.6.2. Oxidation e fermentation test (H and L test)
Isolates were subjected to oxidation fermentation test and or-
ganism was positive. The Vibrio sp was positive in the presence of
oxygen and the results was shown in Fig. 3b.
3.6.3. Indole production test
The isolates were subjected to iodole utilization test and only
photosynthetic bacteria were found to be negative and the result
was shown in Fig. 3c. No red ring formation on the upper surface of
nutrient broth. Yellow color was formed.
(1)
3.6.4. Methyl red test
It is tested for the presence of enzyme to produce acetone or
acetyl methyl carbinol and the isolates shows negative result. The
 P.R. Yaashikaa et al. / Microbial Pathogenesis 99 (2016) 5e13 9

Fig. 1. Culture plate of isolated Vibrio species in TCBS Agar.

Table 1 Table 3
Colonies of Vibrio species in TCBS Agar. Results of polymer hydrolysis.

S. no Appearance of colonies on TCBS Agar Biochemical test Vibrio cholera Vibrio parahaemolyticus

1 Small, green colonies. Starch Positive Positive

2 Smooth, yellow colonies, 2e3 diameter with opaque center Gelatin Negative Negative
3 Large green colonies. Casein Negative Negative

Table 2 result was shown in Fig. 3e. One isolate developed pink color
Morphological characteristics of Isolated Vibrio sp. indicated as positive reaction and the next isolate indicate negative
Organisms Colony characteristics result.
Size Shape Color Elevation Margin

V. cholera Large Round Yellow Slightly raised Entire 3.6.6. Citrate utilization test
V. parahaemolyticus Large Round Green Slightly raised Entire Isolates were subjected to citrate utilization test. The isolates
utilized citrate in the absence of glucose, the color of the media
changed from green to Prussian blue was positive and the result
was shown in Fig. 3f.
Table 4 shows that results of fermentation reactions of Vibrio sp.

3.7. Respiratory reaction

3.7.1. Catalase test

The isolates were tested for catalases test. In Fig. 4a, it was found
that positive which shown that evolution of free oxygen bubble.

3.7.2. Oxidase test

All the isolates were tested for the presence of respiratory
enzyme, cytochrome oxidase and two isolate were found to be
oxidase positive, which was indicated by the immediate appear-
ance of deep purple color and the results was shown in Fig. 4b.

3.7.3. Urease test

The two isolate were subjected to urease test and was found
negative. This was indicated by the absence of pink color.
Fig. 2. Polymer hydrolysis.

3.7.4. Nitrate reduction test

All the isolates were tested for the ability to reduce nitrate. The
results were confirmed from Fig. 3d.
appearance of immediate red color by all the isolates, on addition of
nitrate reagent I & II in equal proportion to the peptone broth
3.6.5. Vogues Proskauer test indicated a positive reaction.
All the isolates were subjected to Vogues Proskauer test and the Table 5 shows that results of respiratory reaction of Vibrio sp.
10 P.R. Yaashikaa et al. / Microbial Pathogenesis 99 (2016) 5e13

Fig. 3. (a, b, c, d, e, f) Fermentation reactions.

Table 4 Fig. 5a and b. Tables 6a and 6b shows that antibiotic sensitivity of

Results of fermentation reactions. Vibrio sp.
Sl. no Name of the test V. cholera V. parahaemolyticus

K/A A/A 4. Discussion

1 Dextrose Acid only Acid only

2 Sucrose Acid only Acid only
The present study deals about the studies on Vibrio species
3 Lactose Acid only Acid only present in prawn and its preservation techniques at an Environ-
4 Maltose Acid only Acid only mental laboratory, SSN College of Engineering. The prawn sample
5 H2S Alkaline Alkaline (Penaeus monodon) was collected from Kovalam beach market,
6 Indole Alkaline Alkaline
Chennai. The study was undertaken to evaluate the local situation
7 M.R Alkaline Alkaline
8 V.P Acid only Alkaline of Vibrio sp in seafood products and to make recommendations to
9 Citrate Acid only Acid only reduce risk associated with the consumption of seafood. Vibrio sp
10 H & L test Acid only Acid only belong to the family Vibrionaceae, are highly abundant in aquatic
environments. The Quantitative analysis of the study on vibrio
isolation methods, usually entails direct plating on to TCBS agar.
In the present study, the prawn samples were plated on to TCBS
3.8. Antibiotic sensitivity agar and Alkaline Peptone water. Green and yellow color colonies
were obtained. From the result of present investigation, totally two
The isolate from prawn were checked for their sensitivity or different Vibrio species has been isolated from the prawn (seafood).
resistance to four different antibiotics and the result was shown in Other organisms such as Pseudomonas, Bacillus, Flavobacterium etc
 P.R. Yaashikaa et al. / Microbial Pathogenesis 99 (2016) 5e13 11

Fig. 4. (a, b). Respiratory reactions.

Table 5
Result of respiratory reaction.

Biochemical test V. cholera V. parahaemolyticus

Catalase Positive Positive

Oxidase Positive Positive
Urease Negative Negative
Fig. 5. a. Antibiotic sensitivity of Vibrio cholera. b Antibiotic Sensitivity of Vibrio
Nitrate Negative Negative
parahaemolyticus.

are also found. The biochemical analysis was done to study the Table 6a
Antibiotic sensitivity of Vibrio cholerae.
virulence potential and confirmation of vibrio strains. All the 2
isolates of Vibrio sp are gram negative short curved rods and they all Antibiotic Symbol Diameter of zone inhibition (mm) Interference
show darting type of motility. The breakdown of starch was rather Penicillin G P e Resistant
more complicated than that of sugars, because of their extensive Clindamycin CD e Resistant
hydrolysis. In this study, the isolates showed positive for starch Piperacilin PI 21 mm Resistant
hydrolysis. Casein is a milk protein composed of amino acid. The Co-Trimoxazole CO 20 mm Sensitive

hydrolysis of casein was mediated by the enzyme protease, which

clearly demonstrated by a clear envelop surroundings the bacterial
growth [20,21]. Table 6b
Antibiotic sensitivity of Vibrio parahaemolyticus.
The present study shows that the isolates were positive for
casein hydrolysis. Gelatin is a protein which can be attacked by Antibiotic Symbol Diameter of zone inhibition (mm) Interference
microorganisms, results from loss of its property to gel, by the Penicillin G P e Resistant
enzyme gelatinase. Lipid is the organic substances, which was hy- Clindamycin CD e Resistant
drolyzed by the enzyme, lipase. This shows a clear zone around the Piperacilin PI 20 mm Resistant
Co-Trimoxazole CO 24 mm Sensitive
growth. All the 2 isolates were positive for lipid hydrolysis.
V. cholerae and V. parahaemolyticus were fermented with sucrose,
dextrose, maltose and lactose which gives positive result for su-
crose, dextrose and maltose and gives negative result for lactose. reactions for methyl red, Voges Proskauer and Citrate utilization
Indole is a putrefactive compound produced by the degradation of test. Present study reveals that, both the isolate showed positive
the amino acid, tryptophan. In the present study, the Vibrio sp were reaction by giving red color, when the indicator was added. Both
found positive for indole, which shows the ability of organisms to the isolate was positive for VP test and the isolate utilized citrate
degrade the amino acid to indole. The Vibrio strains show varying i.e., positive for citrate utilization. All the 2 isolates were positive for
12 P.R. Yaashikaa et al. / Microbial Pathogenesis 99 (2016) 5e13

Table 7a such as cuts, open wounds and abrasions to aquatic environments

Growth of Vibrios at different Temperature. and marine animals. V. cholerae and Vibrio parahemolyticus is an
Temperature (
C) V. cholera (620 nm) V. parahaemolyticus (620 nm) important food borne pathogen. As the isolates showed antibiotic
0 0.007 0.021
resistance, it is a threat to public health as the antibiotic resistant
37 1.096 1.072 determinacies may be transferred to other bacteria of the clinical
50 0.188 0.325 significance. V. cholerae and V. parahemolyticus is a candidate
70 0.233 0.041 vehicle for such transfer because of its diversity and also it can
survive in the gastrointestinal tracts both human and animals.
Rapid development of antibiotic resistance in bacteria and emer-
Table 7b gence of drug resistant microbial disease in aquaculture industries
Growth of Vibrios at different pH.
possess serious problems in environmental, economic and man-
pH V. cholerae V. parahaemolyticus agement and in addition create human health hazards. Survival
3 0.153 0.145 times of Vibrio sp are dependent on factors such as osmotic pres-
5 1.112 1.437 sure, salinity, temperature, pH, moisture content, salt and
7 1.362 1.310
9 0.646 0.568

Table 7c
Growth of Vibrios at different salinity.

NaCl (gm) V. cholera V. parahaemolyticus

0.5 0.056 0.054

1 0.072 0.054
2 0.076 0.072
3 0.725 1.022

catalase, oxidase and nitrate reduction tests and were negative for
urease and H2S test. In the Hugh and Leifson test, all the isolates
showed fermentative reactions. The utilization of amino acids like
arginine and methionine can be determined by the decarboxylation
test. In the study, all the isolates were positive. Antibiotic sensitive
pattern was studied all the 2 isolates showed 100% resistance to-
wards Penicillin G, Clindamycin and Piperacilin. But the isolate
showed 100% sensitive to Co-Trimoxazole.
The effect of various parameters such as temperature, pH, and
salinity were studied and to identify the growth rate of Vibrio sp
and the results were shown in Tables 7a, 7b & 7c. To adjust the
various temperature like 0
C, 37
C, 50
C, and 70
C and keep it for
incubation and read the odd value at 620 nm. The maximum
growth was present in 37
C (Fig. 6a). For pH analysis, keep the
sample at different pH. The Vibrio sp grows best at alkaline pH. So
the maximum growth is present in the pH is 9 (Fig. 6b). At different
salinity the maximum growth of Vibrio sp was studied. According to
increase in the concentration of NaCl the growth also increased
(Fig. 6c). Different preservation method also carried out by
decreasing the growth of Vibrio sp and the results was shown in
Tables 8a, 8b, 8c & 8d. The organic acid used for the preservation
method is acetic acid, oxalic acid, oleic acid. Different concentration
of osmotic pressure is also used for the prevention of Vibrio sp.
Radiation is another preservation method to reduce the growth of
the Vibrio sp. Drying is also applied by the help of direct sunlight,
this was also reduced the growth of the organism. Few days after
the preservation, the growth of Vibrio sp were reduced.

5. Conclusion

Bacterial diseases are one of the major problems affecting pro-

duction, development and expansion of aqua culture. Most of the
bacterial diseases are associated with changes or deterioration of
the aquatic environment. Vibrio sp are widespread in marine and
estuarine environments and several pathogenic species are known
to be commonly associated with outbreaks of vibrio infections due
to consumption of food and water contaminated with human feces
or sewage, raw fish and sea food or with exposure of skin lesion Fig. 6. a. Effect of Temperature. b. Effect of pH. c. Effect of salinity.
 P.R. Yaashikaa et al. / Microbial Pathogenesis 99 (2016) 5e13 13

Table 8a
Preservation by organic acid methods.

Organic 1st day 2nd day 3rdday 4th day 5th day 6th day 7th day 8th day
acids

Acetic acid Green color colony No growth e e e e - -

present
Oleic acid Rough green color Green colony Green colony Green and Yellow colony Yellow colony Yellow colony Yellow colony No,
colony present present present present present present colony
Oxalic acid No growth e - - - - - -

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