Nghiên cứu đặc điểm di truyền và tính kháng thuốc của Vibrio cholerae phân lập tại tỉnh Trà Vinh

MINISTRY OF EDUCATION
CAN THO UNIVERSITY

NGUYEN THI ĐAU

STUDY ON GENETIC CHARACTERISTICS AND
ANTIBIOTIC RESISTANCE OF VIBRIO CHOLERAE
ISOLATES IN TRA VINH
SUMMARY OF DOCTORATE THESIS
MICROBIOLOGY
CODE: 62 42 01 07

CAN THO, 2015


Thesis was completed at CanTho University

Instructor: Assoc. Prof. Dr. Ho Thi Viet Thu

The Doctoral thesis was defended at the Can Tho University.
Time:…………………Date:…………………

This thesis can be found at:
1. National Library of Vietnam
2. Learning Resource Centre of Can Tho University


Chapter 1: INTRODUCTION
1.1 Imperative of the Subject
Vibrio cholerae is a Gram-negative bacteria, the agent of cholera in
humans, causing acute diarrhea and dehydration, diseases occur with other

forms of local epidemic and the pandemic. The world through seven cholera
pandemics, from 1816 to 1923, there were 6 pandemic occurred, these
pandemics started from India caused by V. cholerae O1, the classical biotype .
The 7th pandemic was different from the previous 6 one, it caused by V.
cholerae El Tor, and was derived from the Indonesian island of Celebes in
1961. This was the longes pandemic and it had a greater of influence than the
previous 6 pandemic. Until now, many countries notify the cholera outbreak
caused by this case (Hayes, 2005).
The Mekong Delta, up to 19/08/2010, has 4 localities include Ben Tre,
Tien Giang, Can Tho city and An Giang appear in patients with diarrhea
caused by V. cholerae (Nguyen Hoang Vu, 2011). Tra Vinh Province
geography with numerous potential risks of cholera due to shoreline
stretching for 65 km and in the province of Tra Vinh has major river
systems with a total length of 578 km, including large rivers: the Hau, Co
Chien and Mang Thit river, so it's easy for the circulation of Vibrio
cholerae from Chien river adjacent to Ben Tre province, where the disease
has occurred in 2010.
Many of antibiotics used to treat cholera are resistant to bacteria,
including V. cholerae. This is a concern for public health. Chromosome is
proven the genetic factors of antibiotic resistance genes in bacteria. The
acquisition and transmission of antibiotic resistance genes is due to genetic
factors, such as plasmids, integrons and transposons (Ghosh et al., 2011).
One of the genetic factors is integrated in the chromosome and transmit
antibiotic resistance genes between species in the environment (Burrus et
al., 2004).
Therefore "The study of genetic characteristics and drug resistance of
V. cholerae isolates in Tra Vinh", helping medical deparments with
strategically proper use of antibiotics to reduce the mortality rate, reducing
the cost of treatment.
1.2 Objective: Determine the infection rates and serotype of V.

cholerae on the isolated sample; determine the antibiotic
characteristics and types of antibiotic resistance gene; assess the
1


genetic relationships between the isolates strains and announced
strains and immune response on rabbits with the current vaccine.
1.3 The significance of Thesis: Identification of antibiotic
susceptible to Vibrio cholerae to help Medical departments choosing
the effective Cholera antibiotic treatment in humans.
The thesis result is the scientific basis for the selection of mutated
bacteria to produce cholera vaccine in humans; provide information to help
warn of pathogenicity of Vibrio spp. in water, seafood in Tra Vinh
province.
1.4 New point of thesis
As the first projects in Mekong Delta isolated 6 strains of V. cholerae
from seafood, river and shrimp ponds; identify the type of V. cholerae
serotype: Ogawa and Inaba; identify the similarities in nucleotide
sequences of isolatied V. cholerae strains with the nucleotide sequences of
the V. cholerae strains in the other Southeast Asia countries ; indentify the
tetracycline resistance gene of isolates V. cholerae strains.
The layout
The thesis consists of 108 pages (excluding annexes), divided into the
following sections: Chapter 1: Introduction (4 pages); Chapter 2:
Overview document (42 pages); Chapter 3: The contents, means and
methods of study (18 pages); Chapter 4: Results and discussion (43 pages);
Chapter 5: Conclusions and suggested (1 pages); Reference (page 16). The
thesis has 35 tables, 40 Figure. Total reference is 180, including 09
Vietnamese, 171 English and 11 references from the Web.
Chapter 2: OVERVIEW

2.1 History of cholera
In 1816, the disease appeared in Europe and the US, in the early 20th
century, there were 6 pandemic cholera in the world. In the 60s, range of
pathogens of cholera have been zoned and until recent years, the disease
mostly occurs in Southeast Asia. 1961, El Tor biotype caused pandemic in
Philippines and begin the 7th pandemic. From then, this bacteria continue
to cause pandemic in Asia, the Middle East, Africa and parts of Europe
(Colwell, 2004).
Since 1991, the ratio of cholera caused by V. cholerae O1 have occurred
in Latin America (Levine, 1991; Ries et al., 1992), by 1992 the disease
2


appeared and spreaded quickly by V. cholerae O139 in South East Asia
(Shimada et al., 1993) and recently outbreak in Africa, due to non-O1
serotype, non-O139, which was also an important cause of diarrhea (Sharma
et al., 1998). The study of molecular evolution V. cholerae O1 from 19911995 in Peru, India, and Thailand showed that V. cholerae O1 has undergone
genetic changes at a relatively high level. These changes are important in
understanding the epidemiology and evolution of V. cholerae (Dalsgaard et
al., 1999). In 12/1992 a large pandemic happened, the bacteria were identified
as V. cholerae O139 Bengal. Genetically, O139 Bengal formed from biotype
El Tor but their antigenic structure also changed. People of all ages (even in
the pandemic area ) can be infected, V. cholerae O139 has caused disease in at
least 11 countries in Southeast Asia to 2005 (Garg et al., 2003 )
In Vietnam, cholera is one of the main reasons causing diarrhea since
the 1850s. In 1885, a cholera outbreak occurred and from 1910-1930,
cholera was reported annually (Nguyen, 1962). In 1964, V. cholerae O1
El Tor biotype caused disease in the South of Vietnam. Historically, the
majority of cholera pandemic appeared in the coastal areas of central and
Southern Vietnam. V. cholerae O1 El Tor biotype rarely seen in North

Vietnam. In 1976, the disease has occurred in the city of Hai Phong and
Quang Ninh (Dalsgaard et al., 1999). From 2007 to 2008 and 2010, V.
cholerae O139 isolated from 7 water, named V. cholerae O139. Despite
the rapid spread of O139 in the Southeast Asian region but in Vietnam,
there is very little information about cholera caused by O139 (Dong Tu
Nguyen, 2012).
2.2 Classification - Characteristics of Vibrio cholerae
2.2.1 Classification V. cholerae
V. cholerae is a Gram-negative bacterium that causes cholera in
humans, genus Vibrio and Gammaproteobacteria class. V. cholerae has
two main biotype, the classical and biotype El Tor, and a group of other
serotypes. V. cholerae is classified based on O antigen in the body and the
serum group, so far it has been reported that at least 200 serogroups (Kaper
et al., 1995). Before 1992, the serogroup O1 was the only cause
pandemics. From 1992, serogroup O139 caused outbreak pandemic in
India and Bangladesh. Currently, these 2 serogroup causes cholera
circulation and epidemic; the other V. cholerae serogroup does not cause
the epidemic are pooled into groups of V. cholerae non-O1 and non-O139.
V. cholerae O1 is also divided into three serotypes, Ogawa, Inaba and
Hikojima; these serotypes are divided into 3 types of antigens: A, B and C.
3


2.2.2 Characteristics of Vibrio cholerae
V. cholerae bacterium, also called V. cholerae or cholera, 1μm to 3μm
and 0.5 μm to 0.8μm width. They have 1 flagellum at one end to help
them move very fast wobbly spirals. The cholera bacterium has two major
antigens: H antigen (flagellar) and O antigens from the bacteria body
(somatic O antigen).

Figure 2.1: V. cholerae structure (Korinfo, 2000)
2.2.3 Genetic Characterization of virulence of Vibrio cholerae
The presence of TCP and cholera toxin is controlled by genetic factors
include toxR, Issuer, tcpP, and toxT. All these factors combined into this
virulence characteristic of V. cholerae, this characteristics related to
mobility, the ability to locate the intestine, and toxin production. When V.
cholerae was found in the stool of patients, show that the genetic copy of
V. cholerae is highly toxic and very contagious. The evolution of
pathogens V. cholerae has 2 important stages: firstly, the V. cholerae
strains receive phage TCP and turn into V. cholerae TCP+. After
becoming TCP +, which means fringed bacteria,these serve as receptors
for phage CTXΦ to get into bacterial, and attach its DNA into the
chromosome of V. cholerae phage (lysogenic).
The nature of these CTX and TCP gene are bacteriophage from outside
attached to the chromosome of V. cholerae. V. cholerae is actually
"gentle" bacteria but when they are infected by bacteriophage, they
become toxins and cause disease. V. cholerae strains become toxins
(toxicogenic V. cholerae) and cause disease when they have pili help
bacteria stick to the intestinal mucosa.

4


Figure 2.2: The formation of toxins V. cholerae (Blake, 1994)
2.2.4 The virulence factors
V. cholerae uses two virulence factors, virulence of pili/rod (TCP) and
cholera toxin CTX. TCP, encode of pathogenic factor, a protein from pilus
(Kirn et al., 2000), TCP is also essential for the formation of V. cholerae
colonies located in the small intestine of newborn rats (Taylor et al., 1987)
and human (Herrington et al., 1988). When the formation of colonies in

the small intestine success, V. cholerae secretes toxins that cause cholera.
The toxin stimulates the intestine epithelial cells secrete fluid inside the
small intestine, from which cause diarrheal dehydration. Therefore,
mutations in flagella of some strains of V. cholerae will affect virulence
factors TCP, Figure below show the variation of a flagella.

r
a. Long-Flagella

b. Non-Flagella

c. Short-Flagella

a. Wild strains;
b. Mutant Bacteria;
c. Mutant Bacteria
Figure 2.3: The Flagella -mutant bacteria (Ewen, 2008)
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Bacteria with mutant-gene will lead to defects in flagellar. Through
this Figure, for wild species Figure (2.3a), do not carry the mutant-gene so
the length of flagellum is easily seen through electron microscope; picture
Figure (2.3b) bacteria carry mutant-gene flgT do not formed flagellum;
Figure (2.3c) short-flagella bacteria caused by mutant-gene flagella fliA.
Chapter 3: CONTENTS, MEANS AND METHODS of
RESEARCH
3.1 Content of research
Isolation, identification Vibrio spp. on the samples in Tra Vinh
province; evaluate the serotype of the isolated V. cholerae bacteria;

analyze the nucleotide sequence of V. cholerae according to the 16S rDNA
gene; determine antibiotic resistance genes V. cholerae isolated;
experiment the isolated V. cholerae strains in rabbits to evaluate the
mutation of V. cholerae and the immune response ability to the current
vaccine.
The study period from January 10/2012 - 4/2014 at the College of
Agriculture and Applied Biology Can Tho University; Hospital Central
Can Tho; Research and Development Institute for Biotechnology Can Tho
University; School of Agriculture - Aquaculture Tra Vinh University;
samples sequenced at Macgrogen Korean company.
3.2 Means and methods of research
3.2.1 Means of research
3.2.1.1 Chemical, bacterial culture medium
Distilled water, saline (0.9% NaCl), reagent Kowacs, alcohol 960, 700,
alkaline saline peptone water (ASPW) with concentrations from 0-10%;
Cary- Blair (India); TCBS: thiosulfate-citrate-Thach Bile-salt-sucrose
(Merck); SNA: Saline Nutrient Agar (Merck); MHA: Muller Hinton Agar,
antibiotic discs (Nam Khoa., HCMC).
Biochemical test: oxidase, ONPG, TSI agar (triple sugar iron agar
Saline), ADH (Arginine Dihydrolase), Tryptophan saline; IDS 14GNR,
Nam Khoa, HCMC). 8 disk type antibiotic: streptomycin (10μg),
norfloxacin (10μg), ampicillin (10μg), tetracycline (30μg), azithromycin
(30μg), amoxicillin-clavulanic acid (30μg), trimethoprim-sulfamethoxazole
(25μg) and vancomycin (30μg).

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3.2.1.2 Chemical and biological for PCR
Identifying bacteria based on 16S rRNA gene segments

Table 3.1: Nucleotide sequences of primers for PCR-based 16S rDNA
gene.
Nucleotide sequences of primers (5'3')

Primers

Length (bp)

forward primer // reverse primer

27F

AGAGTTTGATCCTGGCTC’

1492R

TACGGTTACCTTGTTACGACT

ctxA-F

CTCAGACGGGATTTGTTAGGCACG
TCTATCTCTGTAGCCCCTATTACG

ctxA-R
O139rfb-F

AGCCTCTTTATTACGGGTGG

O139rfb-R

GTCAAACCCGATCGTAAAGG

1500
302
449

(1) Weisburg et al., (1991); (2)-(3): Alam et al., (2006)
Determination of antibiotic resistance genes
Reagents for DNA extraction: Tris-HCl, EDTA, H20; chemical PCR
Buffer, MgCl2, dNTPS, DMSO, Taq polymerase and primers determine
antibiotic resistance genes.
Table 3.2: Nucleotide sequences of primers in PCR determine
antibiotic resistance genes.
Antibiotic
group
β-Lactam

Aminoglycosid

Tested
Gene
blaSHV
aac(3)IV

Tetracycline

tetA

Trimethoprim

dhfrI

Nucleotide sequences of primers
(5'3')

Length
(bp)

forward primer // reverse primer

TCGCCTGTGTATTATCTCCC
CGCAGATAAATCACCACAATG
GTGTGCTGCTGGTCCACAGC
AGTTGACCCAGGGCTGTCGC
GTGAAACCC AACATACCCC
GAAGGCAAGCAGGATGTAG
AAGAATGGAGTTATCGGGAATG
GGGTAAAAACTGGCCTAAAATTG

768
286
888
931

(Maynard et al., 2005)
3.2.1.3 Materials Research
160 clam samples collected from Duyen Hai, Cau Ngang district; 100
pig blood samples collected from the slaughterhouse in Tra Vinh City,
Chau Thanh, Duyen Hai, Cau Ngang and Cang Long District; 40 stool
7

samples from patients with diarrhea in hospital of Tra Vinh Province; 150
water samples collected from rivers, the sea and the shrimp farming; 50
shrimp samples collected from Duyen Hai districts. All samples were
stored in cold storage tanks and transferred to the laboratory for isolation.
Oral cholera vaccine (mORCVAX) prepared from the strain of cholera
bacteria including biotype Classical and El Tor biotype and strain of V.
cholerae O139 (Company Limited Vaccine and Biologicals No. 1, Hanoi);
24 New Zealand white rabbits, weight 2-2,5kg.
3.2.1.4 Equipment
Drying cabinet, incubator, refrigerator, autoclave, shaker, sterile
chamber and a microscope, electronic scales, test tubes, petri dishes,
bottles, cylinder, nozzle, slide, pinch, pull, swab wealth repellent, gloves,
alcohol lamps, plastic bags.
3.2.2 Research Methods
3.2.2.1 Methods of isolation, identification of bacteria
* Sampling method
Clam samples: Clam collected at the markets of Cau Ngang and
Duyen Hai District, fresh clams (25gram/piece), cleaned, cut meat (1g),
enrichment in 9 ml of ASPW.
Pig blood samples: Obtained from the slaughterhouse of Tra Vinh
City and the districts of Chau Thanh Duyen Hai, Cau Ngang and Cang
Long . 25 ml blood/ times each facility with 0,5 % salt water, the sample
is transferred to the laboratory, then suck 1ml in 9ml ASPW.
Water samples: Collected on the surface water of shrimp ponds, river,
sea (25 ml each), then take 1ml water in 9ml ASPW.
Shrimp samples: Collected from Duyen Hai districts, fresh shrimp
(150gram / piece), cleaned and took the head, 1 gram sample of finely cut,
extract fluid in 9 ml ASPW.

Stool samples of diarrhea patients: Sterilize swab with sample of
patient's diarrhea stool preserved in Carry-Blair transporting, and
transferred to the laboratory for isolation.
* Method
Samples were cumulative culture twice in the ASPW. 1ml in 9ml
ASPW incubated at 37 0C from 6-8 hours. Then take 1 ml above into 9 ml
ASPW for 2nd incubated at 41,5 0C from 16-18 hours; after isolated in
TCBS at 37 0C /24 hours: colonies may have yellow/green colour.
8


Choose typical colonies into Salt Nutrient Agar (SNA), Vibrio spp.
colonies on SNA: Round, smooth, smooth, milky white.
a. Determine V. cholerae by biochemical reactions (processes ISO/
TS 21872-1: 2007)
The important biochemical testing to detect or distinguish Vibrio with
procedures ISO/TS 21872-1: 2007 (E).
Selecting colonies on SNA to test oxidase reaction; check
glucose/lactose; H2S; Indole test and mobility, halophilic of Vibrio spp. at
NaCl concentrations (0%, 2%, 6%, 8% and 10%).
Vibrio is a genus of Gram-negative bacteria, so after gram staining,
bacteria get light pink colour and have short rod shape, then look under
electron microscope.
b. V. cholerae strain identification by automated machine
identifier (Vitex 2 compact- Biomerieux - BVĐK Can Tho).
The principles of identification of microorganisms is colorimetric
method to identify the biochemical properties of microorganisms through a
color change of card. The identity card is coated with chemicals up to 64
wells, the wells have special chemicals consistent with biochemical
properties of various microorganisms.

c. Serological method
The serological method of V. cholerae done by agglutination with 4
types of antiserum Inaba, Ogawa, and O139 and polyclonal antiserum
Ogawa Inaba, O139 (Tran Linh Thuoc, 2009).
d. Determination of Vibrio spp. by PCR
The bacteria isolated was checked by PCR, based on 16S rRNA gene.
PCR products were analyzed on agarose gel 1.5% in TBE 1X buffer at
100V in 90 minutes and taken with Biorad gel Camera UV 2000, Standard
100 bp (Fermentas company).
3.2.2.2 Analysis of 16S RNA gene sequence and establish the
phylogenetic tree.
The PCR product (DNA) 6 sequences of 6 Vibrio strains: V. choleraeNg3, V. cholerae-O3.2, V. cholerae-O1.2, V. cholerae-81V1, V. choleraeN8 and V. cholerae-85V1, was sequenced in the company Macrogen Inc
(South Korea). The sequences were analyzed and read by software
Bio.Edit, then compared with similar nucleotide sequences set on
GenBank and establishing the genetic tree.
9


3.2.2.3 Determine antibiotic resistance of bacteria V. cholerae.
a. Determination of antibiotic resistance by Kirby Bauer method
* Select 08 antibiotic used to treat intestinal diseases, especially
cholera.
After incubation for 18 to 24 hours, measure the diameter of the zone
of inhibition, including the diameter of the disk and read the results of
antibiotics based on standard tables the antibiotic susceptibility of enteric
bacteria (CLSI, 2010).
b. Determine antibiotic resistance of V. cholerae by PCR
(Polymerase Chain Reaction)
After DNA extraction, continued to identify antibiotic resistance genes
by PCR with primers sequences corresponding to the antibiotic resistance

genes include blaSHV, aac (3)-IV, tetA and dhfrI. PCR products were
analyzed on agarose gel 1.5% in TBE 1X buffer at 100V for 90 minutes
and taken with camera Biorad gel UV 2000 Standard 100bp (company
Fermentas).
3.2.2.4 Sequence analysis of antibiotic resistance genes and
establish phylogenetic tree.
The PCR product (DNA) determine antibiotic resistance genes, then
sequenced in the company Macrogen Inc. The sequences were analyzed,
read by software Bio.Edit, compared with nucleotide sequences
homologous on GenBank and establish genetic tree.
3.2.2.5 Experiment of V. cholerae strains isolated and the immune
response in rabbits against cholera vaccine.
Experiment method.
* Virulence and immune response experiment :
12 experimental rabbits given a dose of 1.5 ml cholera vaccine / rabbit,
repeated on day 14th. Surgery after 28 days of oral vaccines, 12 rabbits
without vaccine but the same feeding.
Inject the isolated bacteria (N8, O3.2, O1.2, Ng3, 85V1 và 81V1) to
the intestines of rabbits of un-vaccine rabbit and the same to oral-vaccine
rabbit, then surgery to determine the immune response of these above
experimen. Rabbits are anesthetized for surgery, the small intestine is tied
into 4 sections, each section 10 cm, 2 cm apart. Use 1ml bacteria needle
(containing 1x 105-5 x 107 CFU) on segments intestine, peritoneal cavity
closed, check the fluid at the time of 3, 6, 9, and 16 hours after injection.
10


The targets :
(i) Fluid accumulation (FA): Fluid accumulation in the intestine
segments are determined by the amount of fluid (ml) / length of the

intestines of rabbits (cm).
(ii) The adhesion of bacteria to the intestinal surface : Cut segments
intestine, shaved the intestinal mucosa or fluid in the intestine, then diluted
liquid into decimal (log 10). Results are calculated by the formula: Mi
(CFU/ ml) = Ai x D1 / v (Mi: the number of bacteria in the initial solution;
Who: The average number of colonies / plate; D1: dilution; v: suspension
volume/disk). Prepare the dilution series:
(iii) Rate of bacterial adhesion to intestinal mucosa of rabbit
Percent (%) of Adhesion = 100 x intestine surface bacteria/ intestine
surface bacteria + CFU fluid (Richardson, 1991).
Data processing: Excel: calculate the average value of the prevalence
rate V.cholerae and antibiotic resistance; Software BioEdit: analyzing the
nucleotide sequences; MEGA software (Treeview): drawing tree
genealogical diagram; Minitab version 16.0: analysis of FA and CFU
values.
Chapter 4: RESULTS AND DISCUSSION
4.1 Results of Isolation and identification of Vibrio spp.
4.1.1 Results of Isolation Vibrio spp.
Vibrio spp. can grow in thiosulfate-citrate-bile salts-sucrose agar
(TCBS): yellow or green colonies depending on the species. Overall,
colonies of the species V.cholerae, V. vulnificus; V. fluvialis và V.
alginolyticus are all yellow, but colonies V. paraheamolyticus is blue;
Colony size is different from different species (Tran Linh Thuoc, 2009).
4.1.2 Result of identifying Vibrio spp. by biochemical reactions
Observing biochemical characteristics to distinguish between species
of Vibrio spp. Most are oxidase and catalase producing, fermented sugar:
V. cholerae, V. paraheamolyticus, V. vulnificus and V. alginolyticus do not
ferment lactose, but V. cholerae and V. alginolyticus are capable of
bacteria tryptophan oxidation into metabolic products with indole origin:
including indole, sketole and formed a red complex. (Tran Linh Thuoc,

2009).
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All species such as V. cholerae, V. vulnificus, V. fluvialis and V.
alginolyticus were negative with urea, V. parahaemolyticus are
positive reaction with urea. Vibrio spp. Grow in saline at 0-2%, 2-6%,
2-8% and 2-10%.
Table 4.1: The results of biochemical of Vibrio spp.
Test SH
V.cholerae
Colonies
Oxidase

Yellow
(+)

Vibrio spp.
V.paraheamolyticus
V.vulnificus
Green

Yellow

(+)

(+)

V.fluvialis

V.alginolyticus

Green

Yellow

(+)

(+)

TSI

(+)

Glucose

(+)

(+)

(+)

Lactose

(-)

(-)

(+)

(-)

(-)

Sucrose

+

(-)

(-)

(+)

(+)

LDC

(+)

(+)

(+)

(-)

(+)

Di động

(+)

(+)

(+)

(+)

(+)

ONPG

(+)

(+)

(+)

(+)

Indole

(+)

(-)

(-)

(-)

(+)

Urease

(-)

(+)

(-)

(-)

(-)

PAD

(-)

(-)

(-)

(-)

Citrate

(-)

(-)

(-)

(-)

(+)

V. cholerae bacteria under electron microscope at 5,000 and 10,000

Figure 4.1: Bacteria under the electron microscope at 5,000 and 10,000
12


Figure of V. cholerae bacteria strain O3.2 through electron
microscope curved shape, 1 - 3µm, short-fragella. V. cholerae uses two
virulence factors from pili / pili (TCP) and cholera toxin CTX. TCP is also
essential for the formation of V. cholerae colonies located in the intestine
of rats (Taylor et al., 1987) and human (Herrington et al., 1988).
The bacteria in this study all had short-fragella,it may be due to defects
in flagellar and mutant-gene so the fragella length is invisible through the
electron microscope.
4.1.3 Result of identifying Vibrio spp. by PCR

Figure 4.2: Amplifying the gene segments 16S-27F and 1492Rbp
The electrophoresis result of the above figure shows the 1500 bp
length PCR product, equivalent to the 16S rRNA gene in all strains of
Vibrio spp. 1500 bp length (Williams et al., 1991) including high
conservation areas and present in most branched bacteria but close
relationship. To compare the test results with the biochemical
characteristics of PCR products, the study recorded the species of Vibrio
respectively: V. cholerae from line 1-6 ; V. fluvialis from 7-11; V.

paraheamolyticus from 12-19; V. vulnific from 20-23; V. alginolyticus
from 24-25. Also in this study, unable to detect any strain of Vibrio
carrying the gene O139rfb and strain carrying the cholera toxins gene
CTXA on water samples and seafood.
Thus, the results of PCR detected strains that do not carry the gene of
Vibrio O139rfb and cholera toxin genes CTX on samples from water and
seafoods. This demonstrates Vibrio O139 do not appear in Tra Vinh
province.
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4.1.5 Detection rate of Vibrio spp. isolated
On the clams, have very diverse presence of Vibrio spp., as these
are species that live in salt water, very suitable for Vibrio spp.,
including V. cholerae are 1, 9%. In pig blood, due to the use of water
from the river for use in the process of slaughtering as washing meat,
blood mixing, positive 2% with V. cholerae corresponding with V.
cholerae isolated from river water (Table 4.3). This result is
comparable with the isolation results in Ho Chi Minh City, which rate
1.1% in water samples and 2.2% in food samples; in Ben Tre, t he rate
in water samples was 5.7% (Nguyen Hoang Vu, 2013), because there
was a cholera epidemic in Ben Tre province in 2010.
Table 4.2: Rate of Vibrio spp. infection on samples.
Samples

Vibrio species

(n = 500)
Clam (n = 160)

Number of
samples

Positive
Samples Rate (%)

V. cholerae

160

03

1,9

V. paraheamolyticus

160

03

1,9

V. vulniticus

160

04

2,5

V. fluvialis

160

05

3,1

V. alginolyticus

160

01

0,63

V. cholerae

100

02

2,0

V. paraheamolyticus

100

02

2,0

+ River

V. cholerae

50

01

2,0

+ Sea

V. alginolyticus

50

01

2,0

+ Shrimp pond

V. paraheamolyticus

50

02

4,0

V. paraheamolyticus

50

01

2,0

40

0

0,0

Pig Blood (n = 100)
Water(n = 150)

Shrimp (n = 50)
Stool (n = 40)
Sum

25

In 25 isolated strains including 6 strains of V. cholerae (24%); 8
strains of V. paraheamolyticus (32%); 4 strains of V. vulnificus
(16%); 5 strains of V. fluvialis (20%) and 2 strains of V. alginolyticus
(8%).
Results from the above table shows the isolated rate of V.

paraheamolyticus was highest (32%), they appear on the clams, in the
river, particularly in the shrimp pond with salinity from 6-8% and on
14


shrimp, consistent with their habitat. Clams are also frequently hosts
of V. vulniticus; V. fluvialis and V. alginolyticus.
4.2 Results of serotyping
Results for serological isolated V. cholerae strains by agglutination
showed 100% (6/6) positive strain with polyclonal antisera (Ogawa, Inaba,
O139), of which 50% (3/6) strains of Ogawa and 50% (3/6) of Inaba
strains.
4.3 Results of the homology between species of Vibrio on GenBank
using BLAST.
In this study, the isolated strains were Vibrio spp., including Ng3,
O3.2, O1.2, N8 and O9.1 have similar rates of nucleotide sequences of
gene segments 16S-27F and 1492R of analysis strains with other strains of
V. cholerae was very high.

.
Figure 4.3: Tree performances evolutionary relationship based on 16srDNA of
isolated Vibrio spp. and some reference strains

Figure above shows the isolated strains had the same characteristics as
the strain originated from the environment, showing the strains of Vibrio
15


spp. always have potential of risks and will easily cause the disease by
receiving the gene from the virulent strain of CTX.

4.4 The antibiotic resistance of V. cholerae
4.4.1 The survey result of V. cholerae antibiotic resistance by
Kirby Bauer method (CLSI, 2010).
Table 4.3: The sensitive and antibiotic resistance of V. cholerae
antibiotic
Streptomycin
Norlfoxacin
Ampicillin
Tetracyclin
Azithromycin
Amoxicillinclavulanic axit
Trimethoprimsulfamethoxazole
Vancomycin

code
Sm
Nr
Am
Te
Az
Ac
SXT/Bt
Van

Number
of
sample

Sensitive
samples

Anti
%

Samples

%

6
6
6
6
6
6

3
6
5
4
4
5

50
10 0
83
67
67
83

3

0
1
2
2
1

50
0
17
33
33
17

6

4

67

2

33

6

2

33

4

67

These results showed that V. cholerae strains in this study are highly
sensitive to many antibiotics such as norfloxacin (100%), ampicillin (83%)
and amoxicillin-clavulanic acid (83%). Also, V. cholerae is resistant to
vancomycin (67%), streptomycin (50%), tetracycline (33%) .... This result
is consistent with the studies of Nguyen Thi Xuan Trang and Nguyen
Ngoc Tuan (2012); Tran Huu Dat (2012).
4.4.3 Results of antibiotic resistance of V. cholerae by PCR
M

1 2

tetA (880bp)

900bp

M: standard 100bp, 1: V.cholerae (T1), 2: V. cholerae (T3)
Figure 4.4: Amplifying 16
of gene segment tetAF and testAR


The electrophoresis results on the above Figure shows that the 880bp
length PCR products corresponding to genes segment tetA discovered in 2
V. cholerae strains. Gene tetA has a speciality conservation sequence, so
the PCR products were all V. cholerae T1 and T2. Through research, did
not dicover V. cholerae strains carry antibiotic resistance genes blaSHV,
aac (3) -IV and dhfrI. V. cholerae strains (T1) and V. cholerae strains (T3)
are isolated from the water environment and they all carry genes resistant

to tetracycline, Aminoglycoside group. Thus some strains in the study did
not contain tetracycline resistance gene (tetA), but they are resistant to
tetracycline, may be due to the presence of other genes encoding resistance
to tetracycline as blaSHV, aac (3)-IV and dhfrI. This result is similar to the
results of Dang et al., (2006).
4.4.5 Results of comparing the nucleotide sequences of the strains
of V. cholerae T1, T3 with wild V. cholerae strain N16961 .
The nucleotide position is inserted and loss corresponds to the position
of amino acid changes in the sequence of the V. cholerae strains, from
which inferences about the mutant type isolated V. cholerae strains.
Table 4.4: Comparing the amino acid position of the wild-type V.
cholerae strains N16961 with V. cholerae strains T1
Codon

14
51
52
69
71
74
105
106
121
142
160
161
164
165
166

Nucleotide
changes

Amino
Acid
changes

nucleotide
T1 loss

AGT→CTG
CCT→TGA
TGG→TCA
CGT→TAG
GTT→TAA
A-T→TCA
CA - →CG - TG→-TG GCT→TAG
GGT→TGA
AAA→TGA
GTA→TGA
CTT→TGA
GAA→TGA
TCA→TGA

Ser→Leu
Pro →End
Trp→Ser
Arg→End
Val→End
→ Ser

Loos 1

→
→
Ala→End
Gly →End
Gly →End
Val →End
Leu →End
Glu→End
Ser→End

17

1

Adding
1
2

Mutant type

Replication error
Recombination error
Replication error
Recombination error
Recombination error
Error
Error

Error
Recombination error
Recombination error
Recombination error
Recombination error
Recombination error
Recombination error
Recombination error


When comparing the nucleotide position of wild V. cholerae strain
N16961 with nucleotide position isolated V. cholerae strains T1, found
that V. cholerae strain T1 has the mutation of adding or loss from 1- 3
nucleotides in multiple codons, to change position and alter the amino acid
protein structure. V. cholerae strains T1 has codon ending at 10 positions,
corresponding to 10 amino acid positions, this is a frameshift mutation by
adding or loss 1 or 2 nucleotides, to stop codons, that will stop the
polypeptide synthesis chain and these enzyme activity will be stopped
(Nguyen Hoang Loc, 2007).
Table 4.5: Comparing the nucleotide position of wild V. cholerae
strain N16961 with nucleotide position isolated V. cholerae strains T3
Codon

Nucleotide
changes

Amini
Acid change

Nnucleotide

T3 loss

Mutant type

6
14
17
19
23

TAA→TGA
AGT→CTG
TGA→ATC
ACA→CCC
GAT→CTC

End →End
Ser→Leu
End→Ile
Thr→Pro
Asp→Leu

Ading
Loos 1
Loos 1
Loos 1
Loos 3

Recombination error
Replication error

Error
Replication error
Replication error

24
32
48
52
53
54
55
63
71
74
95
100
106
124
161

TCA→TAC
AAC→ATC
ACA→CGC
TGG→CTA
GAT→GCG
CTA→GCG
AAA→TAT
GGA→GAA
GTT→GAG
A-T→G- AAA →TGA

TT→TGA
GAA → TAA
CA→TAA
TA→TAA

Ser→Tyr
Asn→Met
Thr→Ala
Trp→Leu
Asp→Gly
Leu→Gly
Lys→Tyr
Gly→Glu
Val→Glu
→
Lys →End
Ile→End
Glu→End
Ala→End
Val→End

Loos 3
Loos 1
Loos 1
Loos 1
Loos 3
Loos 3
Loos 2
Loos 1
Loos 2

2

Replication error
Replication error
Replication error
Replication error
Replication error
Replication error
Replication error
Replication error
Replication error
Error
Recombination error
Recombination error
Recombination error
Recombination error
Recombination error

The same with T1, When comparing the nucleotide position of wild V.
cholerae strain N16961 with nucleotide position isolated V. cholerae
strains T3, shows that V. cholerae strain T3 has the mutation of adding or
18


loss from 1- 3 nucleotides in multiple codons, leading to change position
and alter the amino acid protein structure. V.cholerae strains T3 has codon
ending at 6 positions, corresponding to 6 amino acid positions, this is a
frameshift mutation by adding or loss 1 or 2 nucleotides, to stop codons,
that will stop the polypeptide synthesis chain and these enzyme activity
will be stopped (Nguyen Hoang Loc, 2007).

4.4.6 Genetic relationship of V. cholerae strains based on antibiotic
resistance genes tetA
In this study, two strains of V. cholerae isolated in Tra Vinh carried
tetracycline antibiotic resistance. Isolated from water and clams with
similar of nucleotide sequence with other strains in Thailand, Japan, China,
Indonesia, Brazil and India, 97% similarity with 10 strains; 96% similarity
with 1 strains and 94% similarity with 4 other strains.

Figure 4.5: Genetic relationship of V. cholerae strains based on antibiotic
resistance genes tetA.
Results of genetic tree also showed that two strains of V. cholerae
isolates from T1 and T3 in Tra Vinh river water carries antibiotic
resistance genes had close ralationship of nucleotide sequences (97%) with
other isolated strains of V. cholerae in Indonesia in 2008; Brazil, 2012;
Haiti in 2010; China in 2008; Bangladesh 2009; Thailand in 2014 and V.
cholerae non-O1 isolates vcmD in Japan in 2005.
19


Thus, 2 strains in this study had antibiotic resistance mechanisms
similar to comparative strains and risk of genetic antibiotic resistance
genes is very high in the V. cholerae strains.
4.5 Experiment of virulent V. cholerae mutant strain and evaluate
of immune response in rabbits.
4.5.1 Evaluation results of V. cholerae mutant strains for rabbits
without cholera vaccine.
4.5.1.1 Fluid accumulation After putting the inoculum in the small
intestine rabbits at doses of 105-107, the amount of secreted fluid is
withdrawn through the following chart:

Fluid (ml)

Time

Figure 4.6: Chart of liquid rate after injection of bacteria in the intestine of
rabbit
Through the results, found that wild V. cholerae N1strain 6961
starting fluid accumulation at the time of 9 hours and 16 hours after
injection of bacteria 1.45 ml - 2.3 ml / cm higher than V. cholerae strains
T1 and V. cholerae strains T3 in this study, that carried the tetracycline
resistance gene when injected into the small intestine of rabbits did not
stimulate the rabbit intestinal mucosa secrete fluid, only secreted from 0.8
- 0.9 ml / cm at 6 hours, then decreased to 0:15 - 0.2 ml / cm at the time of
16 hours. This difference was statistically significant (p <0.05) due to the
wild V.cholerae strain N16961 has flagella, strong mobility, easy adhesion
to the intestinal and stimulates the intestinal mucosa produce CT toxic,
causing diarrhea (Sameer et al., 2014; Taylor et al., 1987).

20


4.5.1.2 Content of V. cholerae adhesion to the intestinal mucosa of
rabbit without cholera vaccine.
The number of bacteria counted at the time: 3, 6, 9 and 16 hours
synthesized through the following chart:
CFU V.cholerae

Time (hours)

Figure 4.7: Chart of V. cholerae adhesion in rabbit intestinal mucosa

Through the above chart, the adhesion of bacteria in the intestinal
mucosa was highest at 6 hours and then decreases at the time of 9 hours
and 16 hours in all bacteria strains. Particularly 2 strain T1, T3, the
amount of bacteria only temporarily adhesive at the time of 6 hours from
5,105 to 65,104, then also decreased significantly at 9 hours, only 4,105 to
35,104 and by the time of 16 hours V. cholerae no adhesion.
In summary, the experiment on rabbits shows that mutan- bacteria
temporarily adhesive at 6 hours, then decreased and lost at the time of 16
hours.
4.5.2 Evaluations of immune response in rabbits with cholera
vaccine
4.5.2.1 Fluid accumulation: After putting the inoculum in the small
intestine rabbits at doses of 105-107, the amount of secreted fluid is
withdrawn through the chart:

21


Fluid (ml/cm)

Time (hours)

Figure 4.8: Fluid chart after injection of bacteria in the small intestine
This result shows that all the V. cholerae strain T1, T3, O1.2 Ng3,
85V1, and 81V1 when injected into the intestine for rabbit with vaccine
(mORCVAX), the fluid accumulation at 3 hours but then decreased at 6, 9
and 16 hours, the amount of liquid is a little, due to the intestinal mucosa
have more antibodies attached to receptors on the surface of bacteria,
hindering bacteria from sticking on the host's intestinal mucosa, intestinal
mucosa thus can not secrete toxins and small intestine epithelial cells can

not secrete fluids (Taylor et al., 1987). This difference was not statistically
significant (p> 0.05). Thus, for all rabbits with cholera vaccine had
antibodies should not secrete fluid in the intestine.
4.5.2.2 Content of V. cholerae a dhesion to the intestinal mucosa
rabbit with cholerae vaccine
The number of bacteria counted at the time: 3, 6, 9 and 16 hours
synthesized through the following chart:
CFU V.cholerae

Time (hours)

Figure 4.9: Chart of V. cholerae adhesion in rabbit intestinal mucosa
22


Through the above chart, the adhesion of bacteria in the intestinal
mucosa was highest at 6 hours and then decreases at the time of 9 hours
and 16 hours in all bacteria strains. Particularly 2 strain T1, T3, the amount
of bacteria only temporarily adhesive at the time of 6 hours from 8,104 to
105, then also decreased significantly at 9 hours, only 12,103 to 20,103 and
by the time of 16 hours there was no V. cholerae adhesion, V. cholerae
proved to be inhibited by antibodies secreted by the intestinal mucosa
So, for all rabbits with cholera vaccine had antibodies so the bacteria
only temporarily adhesion at the time of 6 hours, then no adhesion to the
intestinal mucosa in the time of 16 hours, especially T1va T3 strains had
the least content of bacteria.
Chapter 5: CONCLUSIONS AND SUGGESTIONS
5.1 Conclusions
Isolated 25 strains from seafood, river water, shimp pond water, sea
water and pig's blood in Tra Vinh Province, including 25 strains: 6 strains

of V. cholerae (24%), 8 strains of V. paraheamolyticus (32%), 4 strains of
V. vultificus (16%), 5 strains of V. fluvialis (20%) and 2 strains of V.
alginolyticus (8%).
There are 3 V.cholerae strains were positive for monovalent antisera
Ogawa and 3 strains were positive for monovalent antisera Inaba. No
detection of antisera O139.
100% strains of V. cholerae are sensitive to norfloxacin, 83% to
ampicillin and amoxicillin clavulanic acid. 50% are resistance to
streptomycin, 33% are resistance to tetracycline and trimethoprimsulfamethoxazole. There are 2 strains of V. cholerae in the total of 6
tested strains containing antibiotic resistance genes tetA, encoding
gene for tetracycline.
Rabbits had an immune response to the vaccine antigen components
include serogroups O1.
5.2 Suggestions
Continue to explore the pathogenesis and cross immunity between
Vibrio spp. together; choose the mutant strains for testing vaccine on
experimental animals; Application of molecular biology techniques to
study genetic variation for V. cholerae related to pathogenic for humans.

23