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

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

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).


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;

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


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

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.

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

2009).
11


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


(-)

(-)

Sucrose

+

(-)

(-)

(+)

(+)

LDC

(+)

(+)

(+)

(-)

(+)

Di động


(+)

Urease

(-)

(+)

(-)

(-)

(-)

PAD

(-)

(-)

(-)

(-)

Citrate

(-)

(-)


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)


V. fluvialis

160

05

3,1

V. alginolyticus

160

01

0,63

V. cholerae

100

02

2,0

V. paraheamolyticus

100

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

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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.

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

0
1
2
2
1

50
0
17
33
33
17

6

4

67

2

33

6

2

33

4

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



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

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

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

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:


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

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.

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