EVALUATION OF ANTIBIOTIC RESISTANCE OF GRAM – NEGATIVE BACTERIA ISOLATED FROM DONG NAI RIVER

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Consequently, many aquaculture farms in
Vietnam are highly intensive to increase the yield
per area unit. An important factor that affects
the yield is disease outbreaks, which in turn are
irritated by the high stock density of intensive
farming (Subasinghe et al., 2001; Nguyen and 
Ford, 2010). In attempt to protect farms and
INTRODUCTION

Vietnam is known as a country providing
the large amount of aquatic product with the
third ranked in the world (Tran Van Hoa, 2016).
While many aquatic products are exported, the
demand in domestic markets is also high as a
necessary part of the daily diet of Vietnamese.

EVALUATION OF ANTIBIOTIC RESISTANCE

OF GRAM – NEGATIVE BACTERIA ISOLATED

FROM DONG NAI RIVER

Vo Thi Thu Thao, Pham Thi Hoa

International University - Vietnam National University, Ho Chi Minh city
Email:

ABSTRACT

This study assessed the current status of antibiotic resistance of Gram-negative bacteria on Dong 
Nai–Saigon river. Water samples were collected from 4 different areas on the river including: Nam 
Cat Tien forest, La Nga district (Dong Nai province), Sai Gon river (HCMC) and Can Gio district

(Ho Chi Minh city). Sampling sites are close to large aquaculture areas, ranging from upstream 
to downstream of the river. The results showed that 20 strains of Gram negative bacteria, which 
belong to five bacterial species, were identified: Vibrio sp, Serratia sp Enterobacteriaceae sp, 
Pseudomonas sp and Escherichia coli. Sensitivity to antibiotics was determined with four common 
antibiotics used in aquaculture including Ampicillin, Amoxicillin, Chloramphenicol (CHL), 
Tetracycline (TET) and Kanamycin (KAN). AMP antimicrobial resistance was highest (55%), and 
lowest was TET (25%). Most isolated strains (80%) were resistant to at least one antibiotic, of 
which 3 were resistant to all antibiotics. Antimicrobial resistance rates in the five-isolated species 
have shown greater concern needed for the protection of the environment and human health, 
particularly the antibiotics used in agriculture and aquaculture activities.

Keywords: Dong Nai river, Antibiotic resistance, Gram-negative bacteria, Ampicillin, 
Amoxicillin.

TÓM TẮT

Đề tài này đánh giá về hiện trạng kháng thuốc kháng sinh của các khuẩn Gram âm trên sông 
Đồng Nai – Sài Gòn. Mẫu nước được thu thập từ 4 khu vực khác nhau trên sông Đồng Nai bao 
gồm: Rừng Nam Cát Tiên, huyện La Ngà (tỉnh Đồng Nai), sơng Sài Gịn (Tp. HCM) và huyện 
Cần Giờ (Tp. Hồ Chí Minh). Các vị trí lấy mẫu gần với các khu vực nuôi trồng thủy sản lớn, 
kéo dài từ thượng nguồn cho tới hạ nguồn của sông. Kết quả phân lập được 20 dòng vi khuẩn 
Gram âm, được xác định thuộc 5 loài vi khuẩn: Vibrio sp, Serratia sp, Enterobacteriaceae 
sp, Pseudomonas sp và Escherichia coli. Sự nhạy cảm đối với kháng sinh được xác định theo 
bốn loại thuốc kháng sinh phổ biến sử dụng trong nuôi trồng thuỷ sản bao gồm Ampicillin, 
Amoxicillin, Chloramphenicol (CHL), Tetracycline (TET) và Kanamycin (KAN). Tỷ lệ vi sinh 
vật kháng kháng sinh AMP là cao nhất (55%), và thấp nhất là TET (25%). Hầu hết các chủng 
bị cơ lập (80%) kháng với ít nhất một kháng sinh, trong đó có 3 lồi kháng với tất cả các kháng 
sinh. Tỷ lệ kháng kháng sinh trong năm lồi khuẩn được cơ lập đã cho thấy cần quan tâm nhiều 
hơn nữa trong việc bảo vệ môi trường và sức khoẻ con người, đặc biệt là thuốc kháng sinh sử 
dụng trong nông nghiệp và nuôi trồng thủy sản.

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sulfamethoxazole and sulfadimidine (Pham
Kim Dang et al., 2015). The antibiotic
resistance patterns of bacteria are influenced
by selective pressure; thus, may be different
in other geographical areas and may vary over
time (Parveen et al., 1999). Due to the lack of 
regulation, there is very little information about
antibiotics used in aquaculture for almost rivers
in Vietnam, especially in Ho Chi Minh city and
surrounding regions. That is the reason why
Sai Gon - Dong Nai river, which is known as
the main river system in the area with many
aquatic farms and industrial areas along the
river, is considered the antibiotic residue and
appearance of the drug – resistant bacteria
related to the aquaculture activity (Vietnam
Environment Administration, 2016).

It is known that Dong Nai river system
passes through Ho Chi Minh city and other
provinces and have more than 250 rivers and
streams of over 10km each. The basin system
is reach with copious minerals resources
and covered by tropical forests with a great
biodiversity of tropical ecosystems, which
help to protect the area from flood in the rainy
season and maintain water lever for Dong Nai
river in the dry season. Fast urbanization, high
rate of the population growth and the rapid of

economic development included the industry
and agriculture might make the environment
worsened. In the same way, aquaculture
activities, especially occurring strongly at the
La Nga district, Dong Nai province and Can
Gio district, Ho Chi Minh city, have raised
the questions about the situation of utilizing
antibiotic for the animal and the exuberance of
drugs into the environment through wastewater
or fecal. Therefore, this project aims to study the
situation of Dong Nai river system water and
evaluate the level of antibiotic susceptibility
of isolated gram – negative bacteria from the
water sample collected in the Dong Nai river
at four different selective locations, extended
from upstream to downstream, included Nam
Cat Tien forest, La Nga district (Dong Nai
province), Sai Gon river and Can Gio district
(Ho Chi Minh city).

improve yields, farmers must use antibiotics
to prevent and treat diseases for animals.
Hence, aquaculture productions become one
of the main sources of pollution of veterinary
medicines into the environment (Pruden et 
al., 2013; Rico and Van den Brink, 2014). 
As a result, many water resources have been
rendered unwholesome and dangerous to man
and other living organism systems (Bakare et 
al., 2003).

In additionally, awareness of utilizing
antibiotics in aquatic ecosystems is improving
increase in many countries around the world.
Recently, Vietnam has also published new
regulations to prevent ungoverned antibiotic
usage in domestic aquaculture (Official
Decision No. 2625 / QD-BNN-TY, 21/6/2017).
However, the antibiotic usage surveillance
in livestock and aquaculture in the local
areas is still not effective enough which
causes concerns for antibiotic resistance in
pathogenic microorganism. Bacteria cultured
from the collected water sources were tested
for resistance against antibiotics (Tetracycline,
Ampicillin, Kanamycin and Chloramphenicol)
and effect with short term exposure (Pham Kim
Dang et al.,2015). The resistance was displayed 
against the antibiotics tested among the bacteria
cultured from the water. This may indicate that
the existence of antibiotic residue could enter in
the aquatic systems and cause a potential threat
to ecosystem function and influence on human
health (Costanzo SD et al., 2004). Potential
consequences of antibiotic use in animal feeds
are the development of drug– resistant bacteria,
resistance transfer to pathogenic bacteria, and
reduced efficiency of antibiotic treatment for
animal diseases caused by resistant pathogens
(Frappaolo and Guest, 1986). It is worth

considering that, in many ways, the resistance is
already well promoted in bacterial populations
in human, pest and the environment and also
cause the disease with the resistance gene
(Salyers and Amabile-Cuevas, 1997).

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samplings were collected near waterfront while
the others were directly taken at the middle of
the stream using a bottle tied with a 2-meters
string. Three samples (500ml of water for each
sample) at each place were taken into the sterile
screw – capped bottles to preserve the sample
in cold bag until transported to the laboratory
for analysis. The samples were transported to
laboratory for the isolation procedure within
2-3 hours.

MATERIAL AND METHOD
Sampling

The sampling sites were chosen based on
aquaculture activity along the river, extended
from upstream to downstream. The water
samples were taken at four locations: Nam Cat
Tien forest, Sai Gon river (district 2 and Thu Duc
district), La Nga district and Can Gio district
(Figure 1). At the Nam Cat Tien rorest, the

a) Sai Gon river b) Shrimp farm (Can Gio district)

c) La Nga district d) Nam Cat Tien forest
Figure 1. Pictures of sampling sites on this study
Isolation procedure

A 1-ml portion of water samples was
suspended in 9ml of 0.85% NaCl, and 7 –
fold serial dilutions were prepared in 0.85%
NaCl. Portion (0.1 ml) of each dilution were
inoculated onto Tryptic Soy Agar (TSA) and
MacConkey (Mac) agar plates and incubate
for 18 – 24h at 300C (Ehsan, 2015). After

incubated, each different colony, which based
on the morphologies were recovered from each
plate, was streaked into new medium agar
plate to isolate purify and single colony. TSA

plates were utilizing to evaluate a bacterial
concentration. Each isolated colony was
examined by gram staining and morphology
under microscope to preliminary choose the
gram – negative bacteria.

Microbial Identification

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Result
Sampling

The water samples were taken from four
stations as mentioned in the table 2. Each place

has different characteristic to test distinctive
feature might affect the number of bacteria
even the specificity strain. The samples at first,
second and third stations have nearly the same
pH value with around 6.7 to 7.0. Similarly, the
salinity is zero of the first three place. However,
at the fourth station, the pH value is slightly
higher than the others with the average value
is about 7.5. In that period, the water and the
soil in Can Gio district was still influenced by
salt intrusion. Therefore, the salinity value is
very great, over 10‰. Those disparities in pH
and salinity value could affect the number of
bacteria.

Antibiotic resistance determination

Agar Disk Diffusion Test method was used
with the Mueller Hinton agar (Bauer et al., 
1966). The isolates were tested for sensitivity
to four antibiotics applied with the different
dosage for each (µg/ml): Ampicillin (AMP)
(10), Kanamycin (KAN) (30), Tetracyline (30),
and Chloramphenicol (30). The plates were
incubated 24h at 30oC. The result considered

resistant relied on the sign of the good growth on
antibiotic plate and the zone inhibition table 1.

Table 1. Standard for Antibiotic susceptibility

of bacteria (by diameter of zone of inhibition
– in mm) from Clinical and Laboratory

Stand-ards Institute 2015.

Resistance Intermediate Sensitivity

AMP ≤ 13 14 - 16 ≥17

CHL ≤ 12 13 - 17 ≥ 18

TET ≤ 11 12 - 14 ≥ 15

KAN ≤ 13 14 - 17 ≥ 18

Table 2. Features of the water sampling sites

Sampling Stations Characteristic Station (No.) Water pH Salinity (‰)

16th September 2016 Nam Cat Tien

forest (1) Headwater

1.1 6.76 0

1.2 6.81 0

1.3 6.79 0

16th September 2016 La Nga

district (2) Fish farm

2.1 6.84 0

2.2 6.9 0

2.3 6.91 0

30th September 2016 Sai Gon

river (3) Downstream

3.1 7.01 0

3.2 6.94 0

3.3 6.98 0

30th September 2016 Can Gio

district (4) Shrimp farm

4.1 7.52 14

4.2 7.64 17

Isolation and Identification

After isolation procedure, all the gram

– negative bacteria strains were taken for
microbial identification steps using the
biochemical reactions by IDS 14 GNR Kit
provided by Nam Khoa company. The Kit
uses 14 different biochemical reactions caused

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third places have four isolated gram-negative
microorganism, the other have three strains. In
total, there were 20 purified isolated strains of
gram – negative animalcule at four locations
(Table 4).

station and two at second station (13.04%).
Most of the bacteria were the gram – negative
microorganism (86.96%). Gram – positive
bacteria were eliminated leads to the first station
have five strains of gram – negative bacteria
while the second exist eight strains. Besides, the

Table 3. Ratio of isolated bacteria
Stations

(No.) Isolation number

Bacteria

Gram Positive Gram Negative

No. % No. %

1 6 1 16.67 5 83.33

2 10 2 20 8 80

3 4 0 0 4 100

4 3 0 0 3 100

TOTAL 23 3 13.04 20 86.96

The IDS 14 GNR Kit was used to determine
the microorganism by following strictly the
instruction. The result was listed in Table 4
below.

Table 4. Microbial Identification
Station

(No.) Series (No.) Microbial Identification

1 1 Serratia marcescen

1 2 Vibrio metschinikovii

1 3 Vibrio metschinikovii

1 4 Serratia plymuthica

1 5 Vibrio mimicus

2 6 Vibrio hollisae

2 7 Serratia plymuthica

2 8 Serratia plymuthica

2 9 Vibrio mimicus

2 10 Serratia fonticola

2 11 Serratia marcescen

2 12 Pragia fontium

2 13 Vibrio metschinikovii

3 14 Vibrio vulnificus

3 15 Vibrio metschinikovii

3 16 Escherichia coli

3 17 Vibrio metschinikovii
4 18 Pseudomonas chlororaphis

4 19 Vibrio fluvialis

4 20 Vibrio metschinikovii

Antibiotic sensitivity testing

A total of 20 bacteria colonies were
isolated to purified strains of gram – negative
bacteria. The Kit was used to identify the
microorganism. The final steps were examined
antibiotic susceptibility with four types of drug:
Ampicillin, Chloramphenicol, Kanamycin and
Tetracycline.

Table 5 shows the stations particularly with
the amount of antibiotic resistance strains
isolated from each place. The resultshown15
isolated strains from all sampling locations resist
to at least one type of antibiotic. Particularly, La
Nga Village which was the main farmed fishing,
had the highest number isolated microorganism
(8 strains) accompany five were resistant to
antibiotic. The other places had less the resistant
strains than La Nga District.

Table 5. The number of resistant bacteria 
strains isolated in the stations

Stations Isolates (No.) resistance strains Antibiotic
(No.)
Nam Cat Tien forest

(1) 5 4/5

La Nga district (2) 8 5/8

Sai Gon river (3) 4 4/4

Can Gio district (4) 3 2/3

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resist to all four types antibiotic. There are
seven strains resist to two types of antibiotics.
Moreover, Figure 3 exposes the comparison

among those antibiotics. Four strains of bacteria
were not shown the resistance while three were

Figure 2. Antibiotic resistance of bacterial strains isolated from Saigon – Dong Nai river.
Identification by Kit has been indicated the

genera of species combined with the results of
the antibiotic susceptibility test were illuminated
in Table 6. Among the sampling places, the
highest antibiotic resistance could be seen in
the second and third station. The fourth station
has the lowest resistance, especially only two
types of antibiotic indicates the resistance.

In further comparison, Vibrio sp. appear in all 
stations with the high ratio of resistance. There
was difference that Vibrio sp. was resistant with 
mainly AMP and KAN in the first station, CHL
and TET in the fourth station, but in the second

and the third stations, the resistance appears

with all four types of antibiotic. Serratia sp 
has also shown the resistant in the first and the
second stations with the highest aspect scale
at the second place. The Enterobacriaceae sp. 
existed in the water of second location did not
show the drug – resistant. This was the same
situation with the Pseudomonas sp. in the fourth 
station, did not have the resistant. However, in
the third station, Escherichia coli has resistant 
to all of drug. In general, AMP is the antibiotic
which has the highest resistant bacteria ratio
(55%) – resistance whilst the TET has lowest
ratio of resistant bacteria (25%).

Table 6. Antibiotic resistances levels of isolates in the stations – The percentage of 
the number of antimicrobial bacteria over the total number of bacteria

Bacteria groups Station

(No.) Isolates (No.)

Resistance to antibiotic (%)

AMP CHL KAN TET

Vibrio sp 1 3 67 0 100 0

Serratia sp 1 2 50 50 0 0

Total 1 5 60 20 60 0

Vibrio sp 2 3 50 34 34 67

Serratia sp 2 4 50 75 50 25

Enterobacteriaceae sp 2 1 0 0 0 0

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the disease. In recent years, Ho Chi Minh city is
facing the serious pollution, including the water
source. The wastewater that is discharged from
daily activities of citizens is very big quantity
(1.750.000 m3/day by the report of Vietnam

Meteorological and Hydrology Administration,
2017). Nevertheless, a little part of water raw
is treated before going into canals, rivers or
seas. By the report of Vietnam Environment
Administration in 2016, the concentration of
antibiotic in Saigon – Dong Nai river water
is not high, however those antibiotics have
moved and accumulate mainly in downstream
sediments of Sai Gon and Dong Nai river
basins. The accumulation of those antibiotics in
the river sediment for a long time can cause the
local bacteria gradually resisting to those drugs.
Those things can make the consideration to
human health because the water from Dong Nai
river, especially Sai Gon river (upstream river),
is providing supplies to over 20 million citizens
of Ho Chi Minh city and surrounding regions.

Therefore, the consumption of antibiotic in
either animals and patients need to be restricted
in a threshold allowed to prevent the resistance
to antibiotic of microorganism and save the
people.

In this study, 20 strains of gram – negative
bacteria were isolated, after eliminated three
strains of gram – positive organism at four
locations throughout the Sai Gon - Dong
Nai river. This is a respectable result for the
continuous steps in this project. In theory, among
the four sampling places, only La Nga district
and Can Gio district have the strong aquaculture
activities nearly connect with antibiotics, the
ratio of antibiotic resistant bacteria should be
Bacteria groups Station

(No.) Isolates (No.)

Resistance to antibiotic (%)

AMP CHL KAN TET

Vibrio sp 3 3 67 34 67 34

Escherichia coli 3 1 100 100 100 100

Total 3 4 75 50 75 25

Vibrio sp 4 2 0 100 0 50

Pseudomonas sp 4 1 0 0 0 0

Total 4 3 0 67 0 34

Total 20 55 45 45 25

Discussion

The samples were taken at four locations
with distinct feature leads the bacteria were
isolated differently. Although the pH value
might not affect to the number increasing or
decreasing of the bacteria (Toroglu et al., 2005), 
the salinity could be agent that cause the unlike
microorganism between Can Gio district and
the other places. For instance, at the first and
second station, the genus of the microorganism
is the same of Vibrio sp and Serratia sp, but 
at the fourth station (Can Gio), there are the
Vibrio sp in small number and the existence of 
the Pseudomonas sp. This may the outcome of 
the high value in the salinity. During the high
or low salinity period, species of bacteria could
not disperse to survive either with the difficulty
in reproduction (Mills and Geddes 1980) or
dormant burgeon further (Williams 1985; Brock
et al., 2003).

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bacteria with high resistant to antibiotics may
cause a lot of problems for the population in the
future. There will be further study to research
specific strains by modern technology to detect
exactly the gene of bacteria and also resistance
genes for the scientists to find out the solutions
for every specific problem. Finally, the
utilization of antibiotics for animals and people
should be restricted to prevent the multiple drug
– resistance.

Acknowledgement

This research is funded by International
University, VNU-HCM under grant number
SV2016-BT-12.

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480-489.

</div>

EVALUATION OF ANTIBIOTIC RESISTANCE OF GRAM – NEGATIVE BACTERIA ISOLATED FROM DONG NAI RIVER

<b>EVALUATION OF ANTIBIOTIC RESISTANCE </b>

<b>OF GRAM – NEGATIVE BACTERIA ISOLATED </b>

<b>FROM DONG NAI RIVER</b>

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