American Journal of Pure and Applied Biosciences, 2(3), 39-50, 2020

Publisher homepage: www.universepg.com, ISSN: 2663-6913 (Online) & 2663-6905 (Print)

https://doi.org/10.34104/ajpab.020.039050

American Journal of Pure and Applied Biosciences

Journal homepage: www.universepg.com/journal/ajpab

Isolation, Identification, and Antimicrobial Profiling of Bacteria from

Aquaculture Fishes in Pond Water of Bangladesh
Mohammad Zakerin Abedin1*, Md. Sadiqur Rahman2, Rubait Hasan3, Jamiatul Husna Shathi4, Laila
Jarin5, and Md. Sifat Uz Zaman6
1
Dept. of Microbiology, Khwaja Yunus Ali University, Sirajgonj, Bangladesh; 2Dept. of Microbiology, Aqua Laboratory
Quality Feeds Limited, Mymensingh, Bangladesh; 3&4Dept. of Biochemistry and Biotechnology, Khwaja Yunus Ali
University, Sirajgonj, Bangladesh; 5Dept. of Microbiology, LabAid Medical Centre Gulshan Ltd, Dhaka, Bangladesh; and
6
Dept. of Microbiology, Popular Diagnostic Centre Ltd, Dhaka, Bangladesh
*Correspondence: [email protected] (Mohammad Zakerin Abedin, Assistant Professor, Dept. of Microbiology,
Khwaja Yunus Ali University, Bangladesh)

ABSTRACT
Bacterial diseases are widespread and can be of particular importance in fish farming of Bangladesh. This
investigation was done to assess and compare the bacteria diversities and population in local fresh water pond
fishes. Out of 95 samples, 54(56.9%) were Shing (Heteropneustes fossilis), 14(14.7%) were Pangas (Pangasius
pangasius), 9(9.5%) were Pabda (Ompok spp), 7(7.3%) were Thai Koi, (Anabas testudineus), and 11(11.6%)
were others infected fishes such as Shol (Channa striata), Magur (Clarias spp), Tilapia (Oreochromis
niloticus), and Tengara (Mystus cavasius) fishes. Among 95 infected fishes 84(88.4%) were infected with
pathogenic bacteria and 11(11.6%) were normal flora. There were eight types of different isolated spp with
frequency of occurrence were 36 (42.9%) Aeromonas spp, 15 (17.9%) Pseudomonas spp, 7(8.3%) Vibrio spp, 9
(10.7%) Staphylococcus spp, 7(8.3%) Flavobacterium spp, 7(8.3%) Edwardsiella spp, others were 3(3.6%)
Citobacter spp, and Enterobacter spp respectively. All of the isolated pathogenic bacteria showed 84/84(100%)
resistant to Amoxicillin and 18/84(64.3%) resistant to Erythromycin. All the strains showed sensitive to
Ciprofloxacin, Cotrimoxazole, Enorfloxacin, Doxyciline, Clotetracycline, and Colistin with the frequency of
occurrence were 78/84(92.8%), 76/84 (90.5%), 73/84(86.9%), 67/84(67.9%), 53/84(63.1%) and 52/84(61.9%)
respectively. The physiochemical characteristics of 95 pond water samples were analyzed; the average water
temperature, pH, and ammonia were 27.30C, 7.6 and 0.87mg/L respectively. The significant variation in the
physiochemical parameters like water temperature, pH, ammonia were observed within these five types of ponds
water. Hence, it is important to detect fish diseases, responsible pathogens and other agents for protection of our
water resources.

Keywords: Fresh water fishes, Pathogenic microorganisms, antibiotic sensitivity, physiochemical parameters.

1. INTRODUCTION (A. testudineus), Pabda (Ompok spp), Shol (C. striata),

The fresh water fishes are well known and highly Tengara (M. cavasius), Magur (Clarias spp), and
valuable fish species in Bangladesh. Among them, Tilapia (O. niloticus) are very popular in Asian region.
Shing (H. fossilis), Pangas (P. pangasius), Thai Koi A wide range of bacterial pathogens were associated
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with fishes specially, with the aquaculture fishes in (Oreochromisniloticus), and Tengara (M. cavasius)
ponds. The naturally living in the fish’s habitat is and with their water samples were taken from different
reservoir of the indigenous bacterial pathogens such as 18 Upazila in greater Mymensingh, Netrokona, and
Aeromonas spp and Vibrio spp. There are some factors Sherpur districts in Bangladesh between April 2019 to
that induce bacteria to be pathogens such as poor water December 2019. During the collection of fish samples
quality and physiologically uneven fish which permit and water precautionary measures were maintained to
opportunistic bacterial diseases to win (Austin, 2011; avoid touch and ice box were used to maintain cool
Rahman et al., 2019). chain. The samples were then brought to the laboratory
of the Quality Aqua Laboratory, Quality feeds
A number of pathogenic bacteria are related with fish Limited, Mymensingh.
and shellfish for instance Streptococcus spp,
Aeromonas spp, Vibrio spp, Flabobacterium spp and 2.2. Sample processing and enrichment of bacteria -
others pathogens to cause diseases. The economic Aseptic measures were undertaken during the sampling
losses because of fish mortality are mainly caused by procedure to prevent contamination of the samples.
various pathogenic bacteria and other microorganisms Three types of specimens such as intestine, skin and
in Bangladesh (Md et al., 2014). In the fishes there are gill of infected fishes were collected for
two types of bacteria like indigenous bacteria and non- microbiological test. These specimens were taken in a
indigenous microbes. Staphylococcus aureus, sterile chopping board and then minced properly and
Escherichia coli, Clostridium botulinum, Listeria grinded together. Ten (10) gm of samples were
monocytogenes are some examples of non-indigenous homogenized with 90 milliliters(ml) of freshly
microorganisms and then again, indigenous microbes prepared 0.1% peptone water and 0.1 ml of
incorporate Aeromonas spp., Vibrio spp., Salmonella homogenized sample was inoculated according to
spp, and Pseudomonas spp. In Bangladesh, generally standard methods on to selective media such as:
fresh water fishes are found in little rivers, swamp and Rimler Shotts Medium Base agar (for Aeromonas
canal. Now a day’s commercial fish cultivating in lake spp.), Pseudomonas Base agar (for Pseudomonas spp),
is exceptionally well known. The dangerous microbes Thiosulfate citrate bile salt sucrose (TCBS) agar (for
such as Pseudomonas spp, Aeromonas spp, Vibrio spp.), Tryptic Soy Agar (TSA) for enrichment
Staphylococcus spp, Flavobacterium spp, Citobacter of bacterial isolates, Brain Heart Infusion (BHI) Agar
spp Edwardsiella spp, and Vibrio spp that live in every (for fastidious organisms) and finally incubated at
pond causing perilous bacterial diseases, for example, 37ºC for 24 hours.
ulcer, blade decay and tail spoil of fishes (Uddin et al.,
2017). 2.3. Identification of bacterial pathogens - Suspected
bacterial colonies obtained from different culture
The poor water quality is also a great reason that plates were isolated and then streaked on TSA slants,
consumes off the sludge coat or stresses the fishes MIU medium, Simon citrate agar stant and incubated
making them progressively powerless to bacterial overnight at 370C. The pure isolates were characterized
infection. Therefore, this study was designed with a by bacterial cell morphology, alkaline and acidic
view to assess the occurrence of bacterial infection of reaction, H2S (hydrogen sulfide production) and gas
ponds cultivated fishes and their respective ponds production, motility test, indole production, urease test,
water quality assessment and antibiotic susceptibility oxidase test, catalase test, Methyl Red (MR) test, and
analysis against bacterial pathogens of fishes. Voges Praskaure (VP) test.

2. MATERIALS AND METHODS The Gram staining techniques were performed to

2.1. Collection and transportation of samples - A identify Gram positive and Gram negative bacteria.
total of 95 infected fishes including Shing (H. fossilis), The biochemical tests were carried out to identify the
Pangas (P. pangasius), Thai Koi (A. testudineus), pathogens following Bergey’s manual of
pabda (Ompok spp) and others like Shol Bacteriological classification (John et al., 1998).
(Channastriata), Magur (Clarias spp), Tilapia
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2.4 In-vitro antimicrobial sensitivity test - According 2016. Descriptive statistics and chi-square tests were
to the CLSI guidelines (CLSI, 2015), the Kirby- Bauer done to check the statistical evaluation. The p-value
disc diffusion methods were used to in-vitro that considered as significant was <0.5.
antimicrobial susceptibility tests of all the pathogenic
bacteria isolates. The commonly used antibiotics were: 3. RESULTS:
Amoxicillin (10μg), Ciprofloxacin (5μg), Colistin 3.1. Clinical Signs and post mortem findings - After
(25μg), Clotetracyclin (30μg), Doxyciline (30μg), clinical examination of diseased fishes, several severe
Erythromycin (15μg), Cotrimoxazole (25μg), and damages were observed such as equilibrium loss,
Enrofloxacin (5μg). Aeromonas hydrophila (ATCC hemorrhagic ulcerative lesion, rectal protrusion,
7966), Pseudomonas aeruginosa (ATCC 27853), dropsy, body and tail erosion, reddish discoloration
Staphylococcus aureus (ATCC 25923), Vibrio around the eye and mouth, skin lesions on body
cholerae (ATCC 14035), and Flavobacterium surface, and profuse mucous secretion (Fig 1). In
columnare (ATCC 23463) were used as quality control postmortem examination of diseased fishes, organ
throughout the study for culture and antimicrobial enlargement and congestion in internal organs were
susceptibility testing. The suspected isolated bacterial also appeared.
colonies were taken in sterile PBS (phosphate buffered
saline) water and then adjusted to 0.5 McFarland’s 3.2. Bacteria isolated from different diseased fishes
turbidity standard. The bacterial suspension was spread - The isolated bacterial pathogens were founded from
onto Mueller–Hinton agar (Himedia, India) and then different infected fishes are presented in Table 1. Out
impregnated antibiotic discs (Himedia, India) were of 95 infected fishes samples, 84(88.4%) were infected
placed and incubated at 370C for 24 hours. Around the with pathogenic bacteria and 11(11.6%) were normal
discs, the antibiotic zones of inhibition conformed flora. Out of 84 infected fishes spp, 47(49.5%) were
were estimated in diameter of millimeter (mm).The Shing (H. fossilis), 13(13.7%) were Pangas, 6(6.3%)
zone span was really scaled from the focal point of the were pabda (Ompok spp), 7(7.3%) were Thai Koi (A.
anti-microbial plate as far as possible of the reasonable testudineus), and other 11(11.6%) infected fishes were
zone where microscopic organisms could be seen Shol, Gulsha, Magur, Tilapia, and Tengra fishes.
developing. The interpretation of antibiogram was Among the total isolation of 84 bacterial strains, the
measured in millimeter (mm) of diameters as sensitive, highest number was 36(42.9%) for Aeromonas spp, the
intermediate and resistant as per the producer's second highest for Pseudomonas spp, was 15(17.9%),
guidelines. and the next was 9(10.7%) for Staphylococcus spp. On
the other hand, only 7(8.3%) spp of Vibrio spp.,
2.5 Water physio-chemical parameters - Ponds Flavobacterium spp., Edwardsiella spp and the rest of
water temperature and pH were analyzed by the digital 3(3.7%) Citobacter spp, and Enterobacter spp.
thermometer and digital pH meter (Hanna instruments,
USA) respectively. The ideal value of temperature and 3.3. Biochemical tests for bacterial identification -
pH was 30 - 320 C and 7.0 - 8.5 separately. Ammonia, The identification of the pure bacterial isolates were
nitrate, and nitrite were determined by colorimetric performed by biochemical parameters included
method by using Rapid Hanna Test kit (Hanna alkaline reaction, acidic reaction, H2S (hydrogen
instruments, USA), with a special smallest increment sulfide production) gas production, motility test, indole
range of 0.5 mg/L NH3-N, <10mg/L NO3-N, and < production, urea hydrolysis, catalase test, oxidase
0.2mg/L NO2-N respectively. The total hardness and test, Methyl-Red (MR) test, Voges-Praskaure (VP)
alkalinity were measured by EDTA, phenolphthalein, test are presented in Table 2.
and bromophenol titration by using Rapid Hanna Test
Kit (USA) with a special ideal value of 20-100 mg/L In all of the isolated strains it was observed that only
and 50-160 mg/L separately. one strain was found as Gram positive Cocci and the
rest 7 isolates were found as Gram negative bacteria.
2.6 Statistical analysis of experimental data - Data The biochemical analysis revealed that all of the
obtained were analyzed by SPSS version 20 and Excel strains were catalase negative in our experiment.

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Fig 1: Infected fish samples from farmer’s ponds. (a) Shing (H. fossilis) with hemorrhagic ulcerative lesion, (b)
Pangas (P. pangasius) with dropsy, (c) Pangas (P. pangasius) with rectal protrusion, (d) Pabda (Ompok spp) with
pop eye and reddish discoloration around the eye and mouth, (e) Koi (A. testudineus) with deep haemorrhagic
ulcerative lesion on tail region.

Table 1: Bacteria isolated from different infected fishes.

Fish pathogens Name of fishes: N=95 (%)
Shing Pangas Pabda Koi Other Total isolated
fishes bacterial pathogens
Aeromonas spp 23 2 1 4 6 36 (42.9%)
Pseudomonas spp 13 0 0 1 1 15 (17.9%)
Vibrio spp. 1 0 5 0 1 7 (8.3%)
Staphylococcus spp 5 1 0 2 1 9 (10.7%)
Flavobacterium spp 2 5 0 0 0 7 (8.3%)
Citobacter spp, 2 0 0 0 0 2 (2.4%)
Edwardsella spp 1 5 0 0 1 7 (8.3%)
Enterobacter spp 0 0 0 0 1 1 (1.2%)
Total isolated bacterial 47 (49.5%) 13 (13.7%) 6 (6.3%) 7 (7.3%) 11 (11.6%) 84 (88.4%)
pathogens fishes (%)
Normal flora 7 1 3 0 0 11 (11.6%)
Total isolates 54 (56.9%) 14 (14.7%) 9 (9.5%) 7 (7.3%) 11 (11.6%) 95 (100%)

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Out of eight (8) isolates only four strains namely urea hydrolysis test were negative. Various strains of
Aeromonas spp., Vibrio spp., Citobacter spp., Pseudomonas spp showed motile in motility test,
Edwardsiella spp could ferment only glucose and Simon citrate and catalase were positive. The
peptone catabolized and showed alkaline (red color) carbohydrate utilization presented alkaline reaction in
and acidic (yellow) butt. Only Pseudomonas spp was both in stant and butt and both were red in color.
unable to ferment glucose and expressed all stant and These strains also showed only MR-VP test, H2S and
butt were red in color. There was only one strain of gas production were negative. The strains of Vibrio
Gram positive cocci that was able to ferment glucose, spp showed positive motile, indole, oxidase, and
lactose and sucrose and produced yellow color stant Simon citrate test whereas their gas, H2S, urea
and butt. Flavobacterium spp exhibited little bit production and MR test were negative. Out of the 7
different pattern and that was acidic reaction in slant isolated strains it was found that H2S production by
and weak positive butt. The isolated bacteria Citobacter spp, urea production by Pseudomonas spp
Aeromonas spp showed alkaline reaction in stant , and Citobacter spp, Simon citrate test by Aeromonas
acidic reaction in butt, gas production, motility, indole spp and VP tests by Aeromonas spp and Vibrio spp
production, oxidase test, MR-VP, Simon citrate test, were variable respectively.
catalase test were positive whereas H2S production,
Table 2: Results of biochemical tests of the isolated bacterial species from infected fishes.
KIA MIU medium
Bacterial

S. citrate
reaction

Oxidase
isolates

Indole
Gram

Stant

Urea
Butt

Mot

MR
Gas

H2S

Cat
VP
Aeromonas spp Gram negative R Y + - + + - + ± + ± +
Pseudomonas spp Gram negative R R - - + - ± + + - - +
Vibrio spp. Gram negative R Y - - + + - + + - ± +
Staphylococcus spp Gram positive Y Y - - + - + - + + + +
Flavobacterium spp Gram negative Y W - - + - - + + + - +
Citobacter spp, Gram negative R Y + ± + + ± - + + - +
Edwardsiella spp. Gram negative R Y + + + + - - - + - +
(+)=Positive; (-)=Negative reaction; (±)=Variable; R=Red (Alkaline reaction);Y=Yellow (Acid reaction); W= Weak
positive, H2S=Hydrogen sulphide (Blackening); MR= Methyl Red, VP= Voges Proskaure, KIA=Kligler Iron agar,
MIU=Motility indole urea test. Cat=Catalase test, Mot=Motility test,

3.4. Susceptibility to antimicrobial agents’ in-vitro 3.5 Physico-chemical characteristic of pond water -
condition - In our experiment, all of the isolated The recorded pond water parameters of 95 sub-surface
pathogenic bacteria were 84/84(100%) resistant to water samples were studied shown in Table 8. The
Amoxicillin and 18/84 (64.3%) resistant to average range of temperature, pH, ammonia, hardness,
Erythromycin. All the strains showed sensitive to alkalinity, nitrate and nitrite varied depending on their
Ciprofloxacin, Cotrimoxazole, Enorfloxacin, Doxy- cultivated fish types.
ciline, Clotetracycline, and Colistin were 78/84
(92.8%), 76/84 (90.5%), 73/84(86.9%), 67/84 (67.9%), All of the fish water samples showed ammonia
53/84(63.1%) and 52/84(61.9%) respectively. Colistin, concentration higher than their normal value and that
Erythromycin and Clotetracycline expressed interme- was <0.5mg/L. Only Pangas fish pond water expressed
diate sensitivity were very few such as 33.3%, 26.2% the average hardness and Nitrite (NO2) were 126.42
and 15.5% respectively presented in Fig 2. Table 3-7 and 0.37 mg/L respectively that was higher than ideal
showed antibiotic sensitivity patterns all of the value 20-100mg/L and <0.2mg/L respectively Table 8.
individual isolates.
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Resistant Iintermediate Sensitive

120
Sensitivity Pattern (%)

100 100
92.8 90.5 86.9
80
67.9 64.3
60 61.9 63.1

40
33.3
21.4 26.2
20 21.4
2.4 15.5 7.2
10.7 9.5 5.9
4.8 4.8 5.9
0 0 3.6
AMX CIP CL CT DO ERY COT ENR

Antibiotics

Fig 2: Antibiotic sensitivity pattern of all isolates. AMX=Amoxycillin, CIP=Ciprofloxacin, CL= Colistin,
CT=Clotetracycline, DO=Doxycycline, Ery= Erythromycine, COT= Cotrimoxazole, ENR= Enorfloxacin.

Table 3: In-vitro Antibiogram profile of Gram negative and Gram positive bacterial isolates from Shing fishes.
No (%)
Isolated bacteria AMX CIP CL CT DO ERY COT ENR
Aeromonas R 23(100) 0(0) 4(17.4) 7(30.4) 3(13.0) 21 (91.3) 22 (95.7) 1(4.3)
hydraphila
(n=23) S 0(0) 22(95.7) 9(39.1) 2(8.7) 6(26.0) 2(8.7) 1(4.3) 2(8.7)
I 0(0) 1(4.3) 10(43.5) 14 (60.9) 14 (60.9) 0(0) 0(0) 20(87)
Pseudomonas R 13(100) 0(0) 0(0) 2(15.4) 1(7.7) 12 (92.3) 1(7.7) 0(0)
(n=13) S 0(0) 13(100) 9(69.2) 8(61.5) 9(69.2) 1(7.7) 12 (92.3) 13(100)
I 0(0) 0(0) 4(30.8) 3(23.1) 3(23.1) 0(0) 0(0) 0(0)
Staphylococcus R 5(100) 0(0) 1(20) 1(20) 2(40) 5(100) 0(0) 0(0)
(n=5) S 0(0) 5(100) 2(40) 3(60) 0(0) 0(0) 5(100) 4(80)
I 0(0) 0(0) 2(40) 1(20) 3(60) 0(0) 0(0) 1(20)
Flavobacterium R 2(100) 0(0) 0(0) 1(50) 0(0) 1(50) 1(50) 0(0)
(n=2) S 0(0) 2(100) 2(100) 1(50) 1(50) 0(0) 1(50) 1(50)
I 0(0) 00(0) 0(0) 0(0) 1(50) 1(50) 0(0) 1(50)
Citrobacter spp. R 2(100) 1(50) 0(0) 0(0) 0(0) 2(100) 0(0) 1(50)
(n=2) S 0(0) 1(50) 2(100) 2(100) 2(100) 0(0) 2(100) 1(50)
I 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0)
Others (n=2, R 2(100) 0(0) 0(0) 0(0) 0(0) 1(50) 1(50) 0(0)
Vibrio spp. & S 0(0) 2(100) 1(50) 2(100) 2(100) 0(0) 1(50) 2(100)
Edwardsella spp)
I 0(0) 0(0) 1(50) 0(0) 0(0) 1(50) 0(0) 0(0)

AMX=Amoxycillin, CIP=Ciprofloxacin, CL=Colistin, CT=Clotetracyclin, DO=Doxycillin, ERT=Erythomycin,

COT=Trimethoprime, ENR=Enrofloxacin, S= Sensitive, R=Resistant, I=Intermediate

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Table 4: In-vitro Antibiogram profile of Gram negative and Gram positive bacterial isolates from Pangas fishes.
No (%)
Isolated AMX CIP CL CT DO ERY COT ENR
bacteria
Edwarsiella R 5(100) 0(0) 0(0) 1(20) 0(0) 3(60) 0(0) 0(0)
(n=5)
S 0(0) 4(80) 3(60) 3(60) 4(40) 0(0) 5(100) 5(100)
I 0(0) 1(20) 2(40) 1(20) 1(20) 2(40) 0(0) 0(0)
Flavobacterium R 5(100) 0(0) 0(0) 0(0) 0(0) 2(40) 0(0) 0(0)
(n=5) S 0(0) 5(100) 4(80) 5(100) 4(40) 3(60) 5(100) 4(40)
I 0(0) 0(0) 1(20) 0(0) 1(20) 0(0) 0(0) 1(20)
Aeromonas R 2(100) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0)
(n=2) S 0(0) 2(100) 1(50) 2(100) 2(100) 0(0) 2(100) 2(100)
I 0(0) 0(0) 1(50) 0(0) 2(100) 2(100) 0(0) 0(0)
Staphylococcus R 1(100) 0(0) 0(0) 1(100) 0(0) 1(100) 0(0) 0(0)
spp (n=1) S 0(0) 0(0) 0(0) 0(0) 1(100) 0(0) 1(100) 1(100)
I 0(0) 1(100) 1(100) 0(0) 0(0) 0(0) 0(0) 0(0)

Table 5: In-vitro Antibiogram profile of Gram negative and Gram positive bacterial isolates from Koi fishes.
No (%)
Isolated AMX CIP CL CT DO ERY COT ENR
bacteria
Aeromonas R 4(100) 1(25) 1(25) 0(0) 2(50) 0(0) 0(0) 2(50)
(n=4) S 0(0) 3(75) 2(50) 2(50) 2(50) 2(50) 4(100) 2(50)
I 0(0) 0(0) 1(25) 2(50) 0(0) 2(50) 0(0) 0(0)
Staphylococcus R 2(100) 0(0) 1(50) 0(0) 0(0) 0(0) 0(0) 0(0)
spp.(n=2) S 0(0) 2(100) 0(0) 1(50) 2(100) 0(0) 1(50) 2(100)
I 0(0) 0(0) 1(50) 1(50) 0(0) 2(100) 1(50) 0(0)
Pseudomonas R 1(100) 0(0) 0(0) 1() 0(0) 0(0) 0(0) 0(0)
(n=1) S 0(0) 1(100) 0(0) 0(0) 0(0) 0(0) 1(100) 1(100)
I 0(0) 0(0) 1(100) 0(0) 1(100) 1(100) 0(0) 0(0)

Table 6: In-vitro Antibiogram profile of Gram negative and Gram positive bacterial isolates from Pabda fishes.
No (%)
Isolated AMX CIP CL CT DO ERY COT ENR
bacteria
Vibrio spp. R 5(100) 0(0) 0(0) 1(20) 0(0) 1(20) 0(0) 0(0)
(n=5) S 0(0) 5(100) 4(80) 4(80) 4(80) 2(40) 5(100) 5(100)
I 0(0) 0(0) 1(20) 0(0) 1(20) 2(40) 0(0) 0(0)
Aeromonas R 1(100) 0(0) 0(0) 1(100) 0(0) 0(0) 0(0) 1(100)
spp.(n=1) S 0(0) 1(100) 1(100) 0(0) 0(0) 0(0) 1(100) 0(0)
I 0(0) 0(0) 0(0) 0(0) 1(100) 1(100) 0(0) 0(0)

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Table 7: In-vitro Antibiogram profile of Gram negative and Gram positive bacterial isolates from other fishes.
No (%)
Isolated AMX CIP CL CT DO ERY COT ENR
bacteria
Aeromonas R 6(100) 1(16.7) 0(0) 2(33.3) 1(16.7) 6(100) 1(16.7) 0(0)
spp. (n=6) S 0(0) 5(83.3) 4(66.7) 3(50) 2(33.3) 0(0) 5(83.3) 6(100)
I 0(0) 0(0) 2(33.3) 1(16.7) 3(50) 0(0) 0(0) 0(0)
Other isolates R 5(100) 0(0) 0(0) 0(0) 1(20) 3(60) 1(20) 1(20)
(n=5) S 0(0) 5(100) 5(100) 4(80) 4(80) 0(0) 4(80) 4(80)
I 0(0) 0(0) 0(0) 1(20) 0(0) 2(40) 0(0) 0(0)

Table 8: Physico-chemical parameters of water samples collected at a time during April to December 2019.

Nitrate [NO3
(Ideal Value:

(Ideal Value:

(Ideal Value:

(Ideal Value:

(Ideal Value:

(Ideal Value:
20-100mg/L)

Nitrite[ NO2
(mg NO3/l)]
Temp (0C)

<0.5mg/L)

<0.2mg/L)
Value:50 -
Alkalinity
Ammonia

<10mg/L)
160mg/L)
Hardness
30-320C)
Infected

samples

7.8-8.5)
Ponds

(Ideal
fishes

PH

Shing fishes ponds water 27.3 7.6 0.87 73.68 94.4 7.24 0.18

Pangas fishes ponds water 26 7.2 0.9 126.42 139.85 10.7 0.37
Pabda fishes ponds water 27.3 7.8 0.88 88.00 127.11 6.88 0.22
Thai Koi fishes ponds 27.0 7.7 1.87 76.28 140.14 7.64 0.18
water
Others fishes ponds water 26.0 7.5 1.17 99.14 126.42 9.14 0.21

4. DISCUSSIONS respectively. A wide range of bacterial species has

Now not only in Bangladesh, but all over the world our been recovered from local fishes in our work including
local fishes have great demands in the global market Aeromonas spp, Pseudomonas spp, Vibrio spp,
due to their high nutritional quality. The cultivation of Staphylococcus spp, Flavobacterium spp,
these fishes in pond water has been badly influenced Edwardsiella spp, with frequency of 36 (42.9%),
by different factors including various bacterial, viral 15(17.9%), 7(8.3%) 9(10.7%) 7(8.3%) and 7(8.3%)
and fungal contagious pathogens (Sharif et al., 2019). respectively and others were 3(3.6%) Citobacter spp
The mortality rate in adult Shing, Pangas, Thai Koi, and Enterobacter spp, similar findings have also been
Pabda and others local fishes like Shol, Magur, Tilapia reported from other districts of Bangladesh (Al-Harbi
and Tengara were noticed in freshwater cultured ponds and Uddin, 2005; Amal et al., 2010; and Marcel et al.,
situated in Bangladesh. A few numbers of bacterial 2013).
species are pathogenic to fishes, which incorporated
Aeromonas spp, Pseudomonas spp, Vibrio spp, Aeromonas spp, Pseudomonas strains and other
Staphylococcus spp, Flavobacterium spp, isolates were identified from aquaculture pond fishes
Edwardsiella spp, Citobacter spp, and Enterobacter reported by Hossian and Chowdhury (2009). In
spp (Shayoet et. al., 2012; Anshary et al., 2014). Bangladesh, detached and distinguished some
Pseudomonas strains from unhealthy cultivated fishes
However, a total of 84 (88.4%) pathogenic bacteria of Bangladesh. The different types of bacterial species
and 11(11.6%) non- pathogenic bacteria were isolated, of the pond water fishes (Shing) were isolated by
among pathogenic bacteria Gram positive cocci and numerous scientists (Shewan, 2000; and Okaeme,
Gram negative bacilli were 9(10.7%) and 75 (89.7%) 2006). Ahmed and Shoreit (2001) also reported that
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Abedin et al., / American Journal of Pure and Applied Biosciences, 2(3), 39-50, 2020
mixed bacterial contaminations occurred by (100%). Moreover, Staphylococcus spp and Citobacter
Aeromonas spp and Pseudomonas spp. Shing (H. spp were 100% resistant to Erythromycin in our study.
fossilis) is most demandable and highly valuable fish The resistant pattern of Erythromycin (91.3%) and
in Bangladesh. Although, the mortality rate of Shing is Cotrimoxazole (95.7%) were found in Aeromonas spp;
very low but its cultivation in pond water is influenced Erythromycin (92.3%) in Pseudomonas spp.
by different factors such as bacterial pathogens
(Shahen et al., 2019). In any case, the clinical Now a day, in Bangladesh, most available cultivated
manifestations were loss of balance, hemorrhages, skin fishes are panagas (P. pangasius). The isolated
sores, body and tail disintegration, mucous discharge, bacteria were Flavobacterium spp 5(38.5%),
and blockage and development with drain of the inside Edwardsella spp 5(38.5%), Aeromonas spp 2(15.4%).
organs which were comparable with the discoveries of The 100% sensitive bacterial isolates were Edwarsiella
Alicia et al. 2005; Khalil et al. 2010; and Mastan, spp to Cotrimoxazole and Enorfloxacin;
2013. However, the predominant pathogenic bacteria Flavobacterium spp to Clotetracyclin, Ciprofloxacin,
isolated from the pond cultured Shing were Gram- and Cotrimoxazole; Aeromonas spp to Clotetracyclin,
negative bacteria (50.0%), as correspondingly Ciprofloxacin, Doxycycline, Cotrimoxazole and
observed by Marcel et al., 2013. Moreover, in Shing Enorfloxacin, and only one strain of Staphylococcus
fish, the isolated pathogenic bacteria were Aeromonas spp to Doxycycline, Cotrimoxazole and Enorfloxacin,
spp 23(48.9%), Pseudomonas spp 13(27.7%), Gram where Edwarsiella spp. showed poor sensitive to
positive cocci Staphylococcus spp 5(10.6%) and other Ciprofloxacin (80%), Colistin (60%), and
pathogens including Vibrio spp, Flavobacteriem spp, Clotetracyclin (60%), and only one strain of
Citobacter spp and Edwardsella spp were 6(12.8%). Flavobacterium spp showed 60% sensitivity to
Aeromonas spp were the highly isolated bacteria in this Enorfloxacin. In this investigation, only one strain of
study. Among the pathogenic bacterial diseases, Edwarsiella spp was observed to 60% resistant to
Aeromonas spp were the major pathogens that induced Erythromycin. The resistant pattern of Staphylococcus
plagues of ulcerative ailment in fish in Southeast Asia spp was 100% against Clotetracyclin and
and different areas of the world (Anyanwu et al., Erythromycin (Islam et al., 2020).
2014).
Thai Koi (A. testudineus) is the most available and
Bacterial isolates of Pseudomonas spp from Shing (H. cheapable fishes in Bangladesh. The pond water
fossilis) were100% susceptible to Enorfloxacin and cultivated Thai Koi fishes were infected with some
Ciprofloxacin, Staphylococcus spp to Ciprofloxacin bacterial pathogens namely by Aeromonas spp were
and Cotrimoxazole, Flavobacterium spp to 4(57.1%) and secondly by Staphylococcus spp were
Ciprofloxacin and Colistin, Citrobacter spp to Colistin, 2(28.6%) in this study. Comparable isolation rates
Clotetracyclin, Doxycycline, Cotrimoxazole and were accounted by Shittu et al. (2009). In our
Vibrio spp and Edwardsella spp to Ciprofloxacin, investigation, Aeromonas spp expressed 100%
Clotetracyclin, Doxycycline, and Enorfloxacin. Highly sensitivity to Cotrimoxazole and 75% to
sensitive to Ciprofloxacin (95.7%) and Cotrimoxazole Ciprofloxacin, whereas Staphylococcus spp were
(92.3%) were observed in Aeromonas spp and 100% sensitive to Ciprofloxacin, Doxycycline and
Pseudomonas spp respectively and poor sensitive to Enorfloxacin, Cotrimoxazole; third one Pseudomonas
Colistin (69.2%), Clotetracyclin (61.5%), Doxycycline spp were 100% sensitive to Ciprofloxacin,
(69.2%) in Pseudomonas spp., and Clotetracyclin Cotrimoxazole and Enorfloxacin. The parameters of
(60%) and Enorfloxacin (80%) in Staphylococcus spp intermediate sensitivity were 100% to Erythromycin in
were found. In our study, the highly intermediate Staphylococcus spp, and Colistin, Doxycycline and
sensitive were to Clotetracyclin (60.9%), Doxycyline Erythromycin were observed in Pseudomonas spp. The
(60.9%), and Enrofloxacin (87.0%) in Aeromonas spp; significant level of resistance from regularly utilized
Doxycyline (60.0%) in Staphylococcus spp was anti-microbials is similar with Hussain et al. (2014)
observed. In our investigation was observed all and Mostafa et al. (2008).
bacterial isolates were highly resistance to amoxicillin
47
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Abedin et al., / American Journal of Pure and Applied Biosciences, 2(3), 39-50, 2020
Vibrio spp (83.3%) were the most predominant Gram also observed in multiple works done by other
negative bacteria and Aeromonas spp (16.3%) were the investigators. This investigation uncovered that alkali
second common isolated bacteria found in Pabda fish impacted the presence of different pathogenic bacterial
(Ompok spp) in our work. In this study, bacterial spp at all of the sampling areas. It is believed that the
isolates Vibrio spp were 100% sensitive to ammonia in ponds water comes from fish excrement
Cotrimoxazole and Enorfloxacin; Aeromonas spp were and exorbitant feed given to the fish.
100% sensitive to Cotrimoxazole and Ciprofloxacin;
Flavobacterium spp were 100% sensitive to Colistin, 5. CONCLUSION
Ciprofloxacin, and Cotrimoxazole. Vibrio spp were This study has clarified that poor water quality and
highly sensitive (80%) to Colistin, Clotetracyclin and bacterial diseases could be a major cause of
Doxycycline , whereas highly intermediate sensitive considerable economic loss to fish farmers in greater
were to only Erythromycin (100%). Our findings are Mymensingh, Bangladesh. A number of bacterial spp
not correspondent with different examinations led by including Aeromonas spp Pseudomonas spp, Vibrio
Simu et al. (2019). spp, Staphylococcus spp, Flavobacterium spp,
Edwardsiella spp, Citobacter spp, and Enterobacter
In our examination of other fish spp, 6 (54.5%) of spp were the major causes of bacterial diseases to
Aeromonas spp were found in other spp of deshi fishes different fish spp. Clinical examination of diseased
such as Shol (C. striata), Magur (Clarias spp), Tilapia fishes indicated that due to bacterial infection some
(O. niloticus), and Tengara (M. cavasius) and the rest severe damages were found in fish bodies such as
5(45.5%) isolates were Pseudomonas spp, Vibrio spp, equilibrium loss, hemorrhagic ulcerative lesion, rectal
Staphylococcus spp, Edwardsella spp, and protrusion, dropsy, body and tail erosion, reddish
Enterobacter spp. In other fish spp such as Shol, discoloration around the eye and mouth, skin lesions
Magur, Tilapia, and Tengara, bacterial isolates mainly on body surface and profuse mucous secretion. The
Aeromonas spp were 100% sensitive to Enorfloxacin presence of antibiotic resistant isolates imposes a
and low sensitive to Ciprofloxacin (83.3%), Colistin serious concern about the drug of choice for treatment
(66.7%), and Cotrimoxazole(83.3%). Other isolates in fishes. A careful consideration should be given
(Citobacter spp, Enterobacter spp) were 100% before deciding the antibiotic for treatment so as to
sensitive to Ciprofloxacin and Colistin, and low prevent the emergence of antibiotic resistance and
sensitive to 80% Clotetracyclin, Doxycycline, properly maintain the physiochemical parameters in
Cotrimoxazole and Enorfloxacin. All of the strains ponds water to reduce the mortality of aquaculture
showed 100% resistant to Amoxycillin. Similar results fishes in ponds water. Moreover, disease prevention of
were recorded by Hussain et al. (2014) and Mostafa et various local fishes should be carried out by employing
al. (2008). better culture practices and health management to
ensure the highest yields and the optimum quality of
The water quality influenced by the encompassing of
the products.
ecological conditions may affect the pond cultured
fishes. This in the end diminishes the insusceptible 6. ACKNOWLEDGEMENTS
status, activating bacterial contamination prompting
We thank Md. Hashmot Ali; Aqua Laboratory Quality
infection episodes (Amal et al., 2015). The fish
feeds Limited for his assistance in the laboratory
wellbeing status and pond cultured environments were
analysis. We would like to special thank Md Rezanur
reflected due to several bacterial flora. Similar study
Rahaman, Department of Biochemistry and
was conducted by Pakingking et al. (2015). The lake
Biotechnology at KYAU for his expert advice and
water’s physiochemical parameters such as ammonia,
encouragement throughout this difficult study.
hardness, alkalinity, nitrate, nitrite, temperature, and
pH were significant in our study area (Ahmad et al.,
7. CONFLICTS OF INTEREST
2018). In this study, every inspecting site had
distinctive characteristics that were related with the The authors announce that there is no conflict of
nearness of microorganisms. This phenomenon was interest with respect to the publication of this article.

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Abedin et al., / American Journal of Pure and Applied Biosciences, 2(3), 39-50, 2020
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Citation: Abedin MZ, Rahman MS, Hasan R, Shathi JH, Jarin L, and Sifat Uz Zaman M. (2020). Isolation,
identification, and antimicrobial profiling of bacteria from aquaculture fishes in pond water of Bangladesh. Am. J.
Pure Appl. Sci., 2(3), 39-50. https://doi.org/10.34104/ajpab.020.039050

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