Research article

Salikan N. A., K. K Yern, Zain, N. A, M, Isolation of Antibiotic Resistant Bacteria from Rivers in Terengganu,
Malaysia. International Journal of Life Sciences and Biotechnology, 2020. 3(2): p. 241- 257.
DOI: 10.38001/ijlsb.711948

Isolation of Antibiotic Resistant Bacteria from Rivers in

Terengganu, Malaysia

Nurul Aqidah Salikan1 , Kam Kar Yern1 and Nor Azimah Mohd Zain1*

ABSTRACT ARTICLE HISTORY

Infectious disease cause by antibiotic resistant bacteria has been more difficult to treat Received
because the ability of these bacteria to avoid the effect of antibiotic medication. 1 April 2020
Nowadays, water environment especially river has become the reservoir for the Accepted
dissemination of antibiotic resistant bacteria due to antibiotic contamination derived from 6 September 2020
human activities. The exposure to antibiotic resistance bacteria in water increase the health
risks associated with the waterborne bacteria in animals and humans. Therefore, this study
was carried out to investigate the presence of antibiotic resistant bacteria from two rivers; KEYWORDS
Ibai River and Terengganu River in Terengganu. A total of 24 bacterial strains were Antibiotic resistance
isolated from both rivers. Antibiotic susceptibility of the bacteria isolates was assayed bacteria, multiple
according to the Kirby-Bauer disk diffusion method using six types of antibiotics such as antibiotic resistance,
ampicillin, rifampicin, gentamicin, tetracycline, ciprofloxacin and chloramphenicol. The river waters,
results of this study indicate that 100% of bacterial strains were resistant to at least two Terengganu
antibiotics tested. The multiple antibiotic resistant (MAR) index of these bacteria was
calculated and 23 bacterial strains show MAR index of more than 20%. Among the 23
isolates, a total of 10 bacterial strains (2A, 2B, 2C, 2I, 3A, 3B, 4A, 4C, 4D and 4E) were
successfully identified as Chromobacterium violaceum, Enterobacter tabaci, Bacillus
wiedmannii, Acinetobacter baumannii, Klebsiella quasipneumoniae subsp.
quasipneumoniae, Chromobacterium aquaticum, Cupriavidus metallidurans, Weeksella
massiliensis, Cupriavidus necator and Acinetobacter nosocomialis. The results from this
study show that both of the rivers are exposed to high level of antibiotics compound which
may lead the bacteria to develop mechanism of antibiotic resistant. This may cause a
serious threat to the public health and environment.

Introduction

River is the most vital freshwater resources for the human and its use for various purposes
such as agricultural, recreational, and also industrial activity [1]. However, uncontrolled and
overuse of antibiotic in clinical setting and also in intensive animal husbandry gives negative
impact to the water environment [2]. These occur due to the release of antibiotics compound
into the aquatic system through the agricultural runoff or municipal wastewater as the
antibiotic is not fully metabolize in the body human and animal treated with antibiotic [3].
The emergence and development of antibiotic resistance gene in bacteria occur due to natural
selection cause by the high levels of antibiotics in the environment [4].

1
Department of Biosciences, Faculty of Science 81310 Johor Bahru, Universiti Teknologi Malaysia
*Corresponding Autor: Nor Azimah Mohd Zain, e-mail: [email protected]

241
These cause the environment to become the reservoir for propagation and proliferation of
antibiotics resistance genes to pathogen through food web and water [5]. As a consequence,
the probability of becoming exposed to antibiotic resistant bacteria outside clinical setting
increases [6]. For instance, people may become exposed through the preparation and
consumption of contaminated food or ingestion of contaminated water. As a result, cases that
involve the antibiotic resistance by organism elevated and become a threat to public life. This
definitely become a threat to human health as antibiotic resistant bacteria will cause serious
infection especially in debilitated patients which may therefore difficult to treat.
Different defense mechanisms have been developed by bacteria to evade themselves from the
effect of antibiotics that was used against them. The genes encoding these defense
mechanisms are located on the chromosome of bacteria or on extrachromosomal plasmids and
are transmitted to the next generation and these known as vertical gene transfer. Meanwhile,
horizontal gene transfer is plasmids that exchanged among bacteria of different taxonomic
affiliation. Horizontal gene transfer by conjugation is common in nature because there will be
more interaction between bacteria especially when the density of bacteria is high [7].
These environmental antibiotics resistance gene could be a reservoir and horizontally
transferred to human-associated bacteria and thus contribute to antibiotic resistance
proliferation. Antibiotics exert a selection in favor of resistant bacteria by killing or inhibiting
growth of susceptible bacteria; resistant bacteria can adapt to environmental conditions and
serve as vectors for the spread of antibiotic resistance depending on their concentrations [8].
Therefore, uncontrolled use of antibiotics may exert selective pressure and resistant pathogen
will encounter more frequently [9]. The main risk for public health is that resistance genes are
transferred from environmental bacteria to human pathogen.
Therefore, this study aimed to isolate and identify the prevalence of antibiotic resistance
bacteria from Ibai River and Terengganu River in Terengganu through biochemical and
molecular tools through comparison with Bergey’s Manual of Determinative Bacteriology.
The rivers may have become the reservoir for spread of these bacterial genes to the water due
to discharge of antibiotics compound from the human activities such as fishery activities.
Through this study, the implication of antibacterial resistance organism in the river water and
the emergence of antibiotic resistance bacteria were evaluated. The risk of the antimicrobial
resistance from river water to the public health and food security were also assessed.

242
Material and Methods

Study site and sampling

The sampling sites were located at two selected rivers such as Ibai River and Terengganu
River in Terengganu (Figure 1). The first sampling site (Point 1) was selected from the Ibai
River, was located near to small aquaculture farm and residential area in Kampung Laut,
Kuala Ibai at the coordinate of (5.280724, 103.141581). Meanwhile, second sampling site
(Point 2) was selected near to industrial area, residential area and restaurants in Kuala Ibai,
Kuala Terengganu. The samples were collected at the coordinate of (5.278680, 103.168602).
Meanwhile, another river was selected as Terengganu River which located in Kuala
Terengganu. The first sampling site (Point 3) with coordinate at (5.279265, 103.084646) was
located near large aquaculture farms and residential area in Kampung Selar Beladau.
Meanwhile, second sampling site (Point 4) with coordinate at (5.328204, 103.125040) was
located near large aquaculture farm and construction site in Pulau Duyong.
Water sample was collected from two rivers at two different sampling points into the 500 mL
sterile bottle. All the samples were kept in an ice box to maintain its temperature at 4 ̊C while
transporting to the laboratory and stored in cold room until further analysis [8].

Fig 1 The study map on sampling locations of a) Ibai River (point 1, point 2) and b) Terengganu River
(point 3, point 4).

243
Isolation of pure bacterial isolates
In order to isolate the pure bacterial colonies, 1 mL of each water samples were diluted with 9
mL of distilled water. Then, the water samples were serially diluted up to 10-4. After that, 100
µL of each dilution was spread on the nutrient agar. The plates were incubated at 37 ̊C for 24
hours. The single colonies of different morphology from the spread plates were subculture by
streaking method for isolation of pure colonies. The pure colonies of the subculture were
characterized based on observation of their morphology. Gram staining was performed to
further characterize the pure colonies into Gram-positive bacteria or Gram-negative bacteria
[9]
Antibiotic susceptibility testing
Kirby-Bauer disk diffusion method was used by following the guidelines established by the
Clinical and Laboratory Standards Institute (CLSI) to determine the antibiotic susceptibility
profile of the bacterial isolates against six types of antibiotics such as gentamicin, ampicillin,
rifampicin, chloramphenicol, tetracycline, and ciprofloxacin [10]. The antibiotics that were
used in this study are broad spectrum antibiotics that could act against larger group of bacteria
including Gram-negative and Gram-positive bacteria [12]. Broad spectrum antibiotics are
normally used in treatment of community- or hospital-acquired infections [13]. Bacterial
inoculum was prepared by transferring the bacteria into 5 mL of sterile nutrient broth. The
turbidity of the suspension was adjusted to 0.5 McFarland standards. Then, the bacterial
isolate in the nutrient broth was inoculated on Mueller-Hinton agar by spreading it using
sterile cotton swab. The plates were seeded uniformly by rubbing the swab on the entire agar
surface and then incubated for 24 hours [6]. After dried, antibiotic impregnated discs were
placed using sterile forceps with appropriate distance; 30 mm apart and 10 mm away from the
edge of the agar plate with bacterial cultures on it. Each of the antibiotics disc have two
different concentrations which are 10 and 30 µg/mL. Then, all the plates were incubated at
37 ̊C for 24 hours. After overnight incubation, zone of inhibition around each disc was
measured based on the interpretive standard of the CLSI used to define the bacterial isolates
as sensitive, intermediate or resistant to the antibiotic evaluated.
Multiple Antibiotic Resistant (MAR) Index Value
The multiple antibiotic resistant (MAR) index for each bacterial isolate against the antibiotics
tested were calculated using the formula. Multiple antibiotic resistance index is calculated by
the ratio of number of resistant antibiotics to which isolate is resistance to total number of
antibiotics to which organism is tested [14]. MAR index is a tool that helps in analyzing
health risk which value equal or lower than 0.20 indicate low dose of antibiotic used while

244
value more than 0.20 indicates high dose of antibiotic used [10]. Bacteria with MAR index of
more than 20% were selected for further analysis. Equation (1) explains the formula used to
calculate the Multiple Antibiotics Resistant (MAR) index of bacteria. Number of antibiotics to
which the bacteria is resistance (a) has to be divided by total number of antibiotics tested to
bacteria (b) to get value of MAR index [11].
MAR Index value =a/bX100 (Equation 1)

Characterization of the bacterial isolates by biochemical test

The bacterial isolates were further characterized by biochemical test as the bacteria have
different physiology from one species to another. The biochemical tests were performed using
the catalase test, indole test, Mac Conkey agar, urease test, mannitol salt agar, citrate test and
oxidase test. The biochemical test results were compared with Bergey’s Manual of
Determinative Bacteriology to predict the possible bacteria species based on their biochemical
activities.

Results and Discussion

Isolation and Characterization of Bacterial Strain from Both Rivers Water Samples
Total of 24 bacterial colonies were successfully isolated from the water samples of the two
rivers. The pure colonies of the subculture were characterized based on observation of their
morphology. From Ibai River, 4 colonies were isolated from upstream (Point 1) and 9
colonies were isolated from downstream (Point 2). Meanwhile, a total of 6 colonies were
isolated from upstream (Point 3) and 5 colonies were isolated from downstream (Point 4) in
Terengganu River. Table 1 shows the morphology characteristics of the bacterial colonies
isolated from Ibai and Terengganu rivers. Table 2 shows the bacterial colonies isolated from
both Ibai and Terengganu rivers with the Gram stains viewed under 100X magnification.
River is required in almost all the activities of mankind such as agricultural, recreational,
irrigation and industrial purposes. However, water quality in many large rivers in many
countries has been deteriorated significantly due to anthropogenic activities in the past few
decades [15].

245
Table 1 The morphology characteristics of the bacterial colonies isolated from Ibai and Terengganu rivers

Ibai River Terengganu River

Opacity Form Elevation Surface Color Opacity Form Elevation Surface Color

Point 1 Point 3

1A Translucent Circular Umbonate Rough Pinkish 3A Translucent Circular Raised Smooth, glistening White

1B Translucent Circular Convex Smooth, glistening Whitish 3B Translucent Circular Convex Rough Pinkish

1C Opaque Circular Flat Smooth, White 3C Translucent Circular Convex Smooth, glistening Yellowish

1D Opaque Circular Flat Smooth, Blue-black 3D Translucent Circular Convex Smooth, glistening Pinkish

Point 2 3E Translucent Circular Raised Smooth, glistening Whitish

2A Opaque Circular Flat Smooth Blue-black 3F Translucent Circular Flat Smooth Color of agar

2B Translucent Circular Raised Smooth, glistening Whitish Point 4

2C Opaque Irregular Flat Smooth White 4A Translucent Circular Raised Smooth Color of agar

2D Translucent Circular Raised Smooth, glistening Yellowish 4B Opaque Circular Umbonate Rough White

2E Translucent Circular Convex Smooth, glistening Color of 4C Translucent Circular Raised Smooth, glistening Yellowish
agar

2F Translucent Irregular Flat Smooth Whitish 4D Translucent Circular Raised Smooth, glistening Pinkish

4E Translucent Circular Flat Smooth, glistening Color of agar

246
Table 2 The bacterial colonies isolated from both Ibai and Terengganu rivers with the Gram stains viewed under 100X magnification
Ibai River Terengganu River

Point 1 Point 2 Point 3 Point 4

Strain Gram Stain Strain Gram Stain Stain Grain Stain Stain Gram Stain

1A 2A 3A 4A

1B 2B 3B 4B

1C 2C 3C 4C

1D 2D 3D 4D

247
2E 3E 4E

2F 3F

2G

2H

2I

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Antibiotic Susceptibility Testing
The result from the antibiotic susceptibility test in Table 3 and Table 4 shows that all the
isolated bacteria were multiple antibiotic resistant (MAR). All of the isolated bacteria show
resistant to at least two antibiotics tested. The isolated bacteria were tested against both 10
µg/ml and 30 µg/ml concentration of all antibiotics tested, in which many of the bacterial
isolates were resistant towards both concentration of antibiotics tested. Most of the bacterial
isolates were highly resistant towards ampicillin, rifampicin, gentamicin, tetracycline and
chloramphenicol. Meanwhile, only one isolated bacteria in Terengganu River was resistant
towards both concentration of ciprofloxacin used. Furthermore, bacterial isolates from Ibai
River shows no resistant towards ciprofloxacin at both concentrations tested. Low or no
resistance of bacteria towards ciprofloxacin may indicate that both rivers were contaminated
with low level of ciprofloxacin. There may be low use of ciprofloxacin from the human
activities in that area [16].
The discharge of municipal and industrial wastewater to the river water may cause to the
presence of antibiotic residual and antibiotic contamination in the river water [17, 18]. This
may lead to the emergence and development of antibiotic resistant bacteria in river water.
Therefore, in this study two rivers water in Terengganu which were Ibai River and
Terengganu River were selected to study for the correlation between the anthropogenic
activities nearby with the emergence of antibiotic resistant bacteria. In Terengganu, river
plays as important asset for the economic resources especially in Terengganu River where
there were many large aquaculture farms across the river owned by the resident as their source
of income. Aquaculture surrounding environment may also become the cause of antibiotic
pollution in river water [19]. Besides, Terengganu River located near to the construction site
and residential area. It was selected because the location was in the area influenced by the
construction site of drawbridge of Terengganu and large aquaculture farms as well as
residential area. Meanwhile, Ibai River was located in Kuala Ibai, a densely populated area. It
was selected because the location was in the urban area that affected by the residential area,
industrial activities and aquaculture farm. Moreover, Ibai river also is connected with small
rivers which cause the flows of water from other location. There is also a small aquaculture
farm own by the resident at the upstream of this river.

249
Table 3 Antibiotic susceptibility tests for the bacterial colonies isolated from Point 1 and Point 2 at Ibai River.

Antibiotics Gentamicin Ampicillin Rifampicin Chloramphenicol Tetracycline Ciprofloxacin

Bacterial 10 30 10 30 10 30 10 30 10 30 10 30
Colonies µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml

1A R R R R R R R S S S S S

1B R S R R R R R R R R S S
Point 1
1C S S R R S S R R R S S S

1D R R R R R S S S S S S S

2A R S R R R S S S S R S S

2B R R R R R R R R R S S S

2C S S R R S S R R S S S S

Ibai 2D R S R R R R R R R R S S

River Point 2 2E R S R R R R R S R S S S

2F S S S S R R R R R S S S

2G R R R R R R R R R S S S

2H S S R R R R R R R R S S

2I R S R R R R R S R S S S

250
Table 4 Antibiotic susceptibility test for the bacterial colonies isolated from Point 3 and Point 4 at Terengganu River.

Antibiotics Gentamicin Ampicillin Rifampicin Chloramphenicol Tetracycline Ciprofloxacin

Bacterial 10 30 10 30 10 30 10 30 10 30 10 30
Colonies µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml

3A R S R R R R R R R R S S

3B R R R R R R R S S S S S

3C R S R R R R R R R R S S
Point 3
3D R S R R R R S R R R S S

3E S R R R R R R R R S R R

3F R S R R S S S S S S S S

Terengganu 4A R S R R R R R S R R S S
River
4B R S R R S R R R R R S S

4C S S S S R S R R R R S S

Point 4 4D R R R R R R R R R S S S

4E R S R R R R R R R R S S

251
MAR Index Analysis
From the Figure 2 (a), a total of 13 isolates from Ibai River shows MAR Index more that
20%. Meanwhile for Terengganu River, 10 out of 11 isolates shows MAR Index more that
20%. Based on the results, all the bacterial isolates were multiple antibiotic resistant (MAR)
as it resistant to at least two antibiotics. MAR index with value more than 0.2 indicate a ‘high-
risk’ source of antibiotic contamination [20]. The location was in the urban area that affected
by the residential area, industrial activities and aquaculture farm. Besides, Ibai River also is
connected with small rivers which cause the flows of water from other location. There is also
a small aquaculture farm own by the resident at the upstream of this river. In this study, the
samples obtained from Terengganu River were collected near to an aquaculture farm,
industrial area, near to construction site area and also from the location that were close to the
residential area. These findings show clear evidence of antibiotic resistant bacteria emergence
at the sampling locations.

MAR INDEX for 10 µg/mL MAR INDEX for 10 µg/mL

MAR INDEX for 30 µg/mL MAR INDEX for 30 µg/mL
90 90

80 80

70 70
MAR INDEX (%)
MAR INDEX (%)

60 60

50 50

40 40

30 30

20 20

10 10

0 0
1A 1B 1C 1D 2A 2B 2C 2D 2E 2F 2G 2H 2I 3A 3B 3C 3D 3E 3F 4A 4B 4C 4D 4E
Antibiotic Resistant Bacteria in Ibai River Antibiotic Resistant Bacteria in Terengganu
River

a) b)

Fig 2 MAR Index for bacteria isolated from a) Ibai River and b) Terengganu River.

Biochemical Test
Table 5 tabulates the biochemical tests were conducted for all the isolates collected from Ibai
and Terengganu rivers. Based on the table, it can be concluded that the different in bacterial
population for different point in the same river may be due to the human activities around the
sampling areas. From the result of Gram staining, there were 2 out of 24 bacterial colonies

252
(8.33%) were Gram-positive with rod shape. Meanwhile, 9 bacterial colonies (37.5%) were
Gram-negative bacteria with cocci shape and 13 bacterial colonies (54.2%) were Gram-
positive bacteria with rod shape. This meant that, most of the isolated bacterial were Gram-
negative bacteria. The pH of both river water which has slightly acidic; range from pH 5 to 6
favor for the Gram-negative bacteria. In Point 1, with total of 4 bacterial colonies, one of them
was Gram-positive bacteria with rod shape, 2 colonies were Gram-negative bacteria with rod
shape and the rest was Gram-negative bacteria with cocci shape. Meanwhile in Point 2, all the
bacterial isolates were Gram-negative bacteria with 5 colonies were rod shape and 4 colonies
were cocci shape. The same to Point 3, most of the bacterial isolates also were Gram-negative
bacteria with 4 colonies were rod shape and 2 colonies were cocci shape. From Point 4, one of
the bacterial isolates was Gram-positive bacteria with rod shape. The rest of the bacterial
isolates were Gram-negative bacteria with 2 colonies were rod shape and 2 colonies were
cocci.

253
 Table 5 Biochemical tests conducted for isolates from Ibai River and Terengganu River.
Ibai River Terengganu River

Point 1 Point 2 Point 3 Point 4

Biochemical Test Biochemical Test Biochemical Test Biochemical Test

Mannitol Salt Agar

Mannitol Salt Agar

Mannitol Salt Agar

Mannitol Salt Agar

Mc Conkey Agar

Mc Conkey Agar

Mc Conkey Agar

Mc Conkey Agar
Conclusion Conclusion
Strain

Strain

Strain

Strain
Catalase

Catalase

Catalase

Catalase
Oxidase

Oxidase
Oxidase

Oxidase
(Family Type) (Family Type)
Urease

Urease

Urease

Urease
Citrate

Citrate

Citrate

Citrate
Indol

Indol

Indol

Indol
Conclusion Conclusion
(Family Type) (Family
Type)

Vibrio sp. Enterobacter Klebsiella sp. Vibrio sp.

sp.
Enterobacter Enterobacter Pseudomon
- + - - - + - sp. + - - - - - - Salmonella sp. + + + - + - + sp. + + - - - + - as sp.
1A

2A

3A

4A
Pseudomonas Pseudomonas Salmonella sp. Proteus sp.
sp. sp.

Pseudomonas Proteus sp. Vibrio sp. Staphylococ

sp. cus sp.
Salmonella sp. Pseudomonas
+ + + - + + + + + + - - - + - + - - - + - + - - - - - -
1B

2B

3B

4B
Vibrio sp. sp. Bacillus sp.
Vibrio sp.
Proteus sp. Proteus sp.

Bacillus sp. Proteus sp. Enterobacter Pseudomon

sp. as sp.
Staphylococcu Pseudomonas
+ - - - + + - s sp. + - + - + + - sp. - - - - - - - Proteus sp. + - - + - + - Enterobacte
1C

2C

3C

4C
r sp.
Klebsiella sp. Pseudomonas
sp. Vibrio sp.

254
 Klebsiella sp. Salmonella sp. Enterobacter Vibrio sp.
sp.
Salmonella sp. Enterobacter Pseudomon
+ - - - + - - + - + - - - + - + - - - - - + + - - - + -
1D

2D

3D

4D
sp. Klebsiella sp. as sp.
Proteus sp.
Klebsiella sp. Vibrio sp. Proteus sp.

Vibrio sp. Vibrio sp. Klebsiella

sp.
Enterobacter Pseudomonas
- + - + - + - sp. + + + - - + + sp. + + + - + - - Proteus sp.

2E

3E

4E
Pseudomonas Proteus sp. Salmonella
sp. sp.

Proteus sp. Salmonella sp.

Salmonella sp. Klebsiella sp.

- - + - - - - + - - - + + -
2F

3F
Enterobacter Proteus sp.
sp.

Klebsiella sp.

Pseudomonas
- + - - - - -
2G

sp.

Salmonella sp.

Proteus sp.

+ + + - + - + Salmonella sp.
2H

Vibrio sp.

Vibrio sp.

+ + + - - - - Salmonella sp.
2I

Proteus sp.

255
Conclusion

This study showed that a total of 24 pure bacterial colonies were successfully isolated from
water samples of two selected rivers which are Ibai River (Point 1: 4 colonies; Point 2: 9
colonies) and Terengganu River (Point 3: 6 colonies; Point 4: 5 colonies) in Terengganu. The
bacterial isolates were tested for its susceptibility towards 6 antibiotics; gentamicin,
ampicillin, rifampicin, chloramphenicol, tetracycline, and ciprofloxacin. All of the isolates
from Ibai River shows MAR index more than 20%. Meanwhile a total of 10 out of 11 isolates
from Terengganu River shows MAR index more than 20%. The results obtained showed that
most of the isolates were multiple antibiotic resistance, indicated that Ibai and Terengganu
rivers might consist of ‘high-risk’ source of antibiotic contamination.

Abbreviations
CLSI: Clinical and Laboratory Standard Institute; MAR: Multiple antibiotics resistance

Acknowledgements
We sincerely thank the staff and administration of the Universiti Teknologi Malaysia for their assistance and
support in this study.
Funding

This study is funded by the Geran Universiti Penyelidikan (GUP) sub vote 17H74 and UTMShine Grant sub vote
04G97, which is supported by the Universiti Teknologi Malaysia.

Availability of data and material

Please contact the corresponding author for any data request.

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