Bangladesh J. Zool.

48(1): 335-346, 2020 ISSN: 0304-9027

eISSN: 2408-8455

SURVEILLANCE OF ESCHERICHIA COLI IN A FISH FARM OF SYLHET,

BANGLADESH

Rizoneul Haq Reza1, Shahena Aktar Shipa1, M. Niamul Naser2

and Md. Faruque Miah*1

Zoology, Fisheries and Marine Biotechnology Research Unit, Department of

Genetic Engineering and Biotechnology, Shahjalal University of Science and
Technology, Sylhet, Bangladesh

Abstract: The study was accomplished to investigate Escherichia coli from two
freshwater fish, Tilapia (Oreochromis niloticus) and Mrigal (Cirrhinus mrigala),
collected from a fish farm in Sylhet, Bangladesh. Six of each fish were analyzed to
isolate and detect Escherichia coli, and E. coli was identified based on
morphological and biochemical characteristics. The antibiogram of E. coli was
investigated in different generations using eight antibiotic discs such as
Chloramphenicol (CH), Streptomycin (S), Gentamycin (G), Ciprofloxacin (CI), Co-
trimethoxazole (CO), Azithromycin (AZI), Erythromycin (E) and Novobiocin (NV),
and the sensitivity of E. coli was found as 100%, 25%, 100%, 75%, 87.5%,
81.25%, 0%, 0% respectively. Among the 8 antibiotics, for Erythromycin (E) and
Novobiocin (NV), the observed resistance pattern of E. coli was 81.25% and 87.5%
respectively, whereas, for the rest of the antibiotics, it was 0%.

Key words: freshwater, aquaculture, Escherichia coli, antibiotic resistance

INTRODUCTION
Bangladesh is rich in fisheries resources and recognized as the third
aquaculture country in the world (FAO 2018), including vast culture potential
where the inland aquaculture sector is contributing more than 55% of the total
production of Bangladesh (DoF 2016). Among 266 freshwater fish including16
exotic species, around 25 species are being cultured (IUCN 2014, FRSS 2016).
Freshwater aquaculture is mainly comprised of pond farming in Bangladesh
where Tilapia (Oreochromis niloticus) and Mrigal (Cirrhinus mrigala) are
commonly cultured species. However, among the other causes, diseases are
responsible for the huge loss of production in aquaculture, especially in
developing countries as they contain 90% of the aquaculture farm. For example,
in Chile, 2 billion dollars lost caused by infectious salmon anemia alone and

*Author for Corresponding: <[email protected]>, 2Department of Zoology, University of Dhaka,

Dhaka 1000, Bangladesh.
©2020 Zoological Society of Bangladesh DOI: https://doi.org/ 10.3329/ bjz.v48i2.52373
336 Reza et al.

15% loss of total fish production to diseases in China (Leung et al. 2013 and
Assefa et al. 2018).
Different pathogenic bacteria are involved to impact the fish production in
Bangladesh whereas few fecal coliforms were observed (Mandal et al. 2009) as a
fish pathogen that often contaminants of food and water through E. coli is
mostly non-pathogenic (Dutta et al. 2010, Soliman et. al. 2010, Eze et al. 2011).
Generally, for the potential sewage pollution, based on strains, E. coli present in
fish are considered as an indicator (Hanson et al. 2008). Inside the intestine of
fish, E. coli commonly resident as non-pathogenic but when expanding outside
the intestine, it can be responsible for causing disease, resulting in entero-
toxigenic (Lee and Marks 2009) whereas 18 toxigenic E. coli were isolated (Vieira
et al. 2001). The sensitivity of different antibiotics was examined and found high
effect against E. coli isolates from fish samples (Sukumaran et al. 2012, Soliman
et al. 2010). The reason for the versatility of E. coli strains of involving different
strains with different diseases is because they obtaining virulence genes of
different sets. E. coli strains are related with different disease-causing fish
virulence genes as well (Teophilo et al. 2002, Gupta et al. 2013).
As a remarkable number of fish species are available in the freshwater of
Bangladesh, tilapia (O. niloticus) and mrigal (C. mrigala) contribute an important
role in nutrition as well as the national economy. Though few researches
observed the occurrence of fecal coliform mostly in E. coli of Nile tilapia
(Thampuran et al. 2005, Mandal et al. 2009). However, information is not
available for mrigal (C. mrigala) in Bangladesh. Due to the high economic value
of these two fishes in Bangladesh, this research is aimed to observe the status of
fecal coliform in a farm condition of Sylhet and to know the surveillance of E. coli
in terms of occurrence, detection, and antibiogram assay.

MATERIAL AND METHODS

Sample collection: The study was conducted in the Department of Genetic
Engineering and Biotechnology (GEB) at Shahjalal University of Science and
Technology (SUST), Sylhet, Bangladesh. Total 12 fish of Tilapia (Oreochromis
niloticus) and Mrigal (Cirrhinus mrigala), 6 for each were collected from a farming
pond of Sylhet, Bangladesh, which then immediately transported to the Zoology,
Fisheries and Marine Biotechnology Research Unit in the Department of GEB,
SUST for further studies. Fish samples were identified according to the
morphometric characteristics (Shafi and Quddus 1982, Rahman 1989) and E.
coli was isolated from the liver, heart, gills, scales surface, and fin of the fish
samples.
Surveillance of Escherichia coli 337

Preparation of experimental materials: The isolation of the bacteria from

experimental fish and identification of E. coli isolates was performed using
morphological and biochemical characteristics. All materials like Petri dishes,
test tubes, stock bottles, etc. were washed with detergent and dried at 70 0C in
an oven drier and sterilized at 1700C for 1.5 hours by a hot air sterilizer. The
tips were washed and autoclaved at 1210C for 15 minutes. Then the glassware
was again put into a drier at 700C before use. Most of the sterilization processes
were followed according to the instructions described by Barrow and Feltham
(1993).
Isolation of E. coli by selective culture media: First of all, 51.53 grams of
MacConkey agar was added to a flask containing 1000 ml of distilled water, and
to dissolve the medium completely, the heat was applied until the boiling.
Through the autoclave, the medium was subjected to sterilization. Furthermore,
38 grams of EMB agar base (Himedia, India) also added to a flask containing
1000 ml of distilled water and for a fine mixture, the heat was applied. Again, for
sterilizing the medium, autoclave was done at 1210C (15 psi) for 15 minutes and
then, to lowering the temperature of the medium, it was placed into a water bath
of 450C. When the medium was solidified, to confirm the sterility, the Petri
dishes were kept incubated at 370C overnight and then stored at 40C in the
refrigerator until used. The samples collected were placed on a MacConkey agar
plate by using a sterile loop and cultured on the medium by the streaked plate
technique, which were then incubated at 370C for 24 hours. The appearance of
the colonies a characteristic green metallic sheen with a dark center was
considered positive for E. coli.
Performance of pure culture of E. coli: About 500 ml of Nutrient Agar (NA)
medium was prepared followed by Cappuccino and Sherman (2007) and stirred
by a magnetic stirrer. Following the previous autoclave procedure, the medium
was sterilized and after reducing the temperature of the medium up to 60˚C, it
was poured into sterile Petri dishes with the amount of 25 ml each, which are
then kept for future use after the solidifying of the plates. A single colony was
taken from EMB agar plate by using sterile loop and inoculated on the NA
medium. The plates were then incubated at 370C for 24 hours.
Morphological Identification of E. coli strains: The bacterial colonies grown on
agar medium were recorded for their colony characteristics such as size, shape,
the color of the colony. Morphological characters of the isolates such as shape
and size were recorded during Gram’s staining procedure with the fresh
subculture of 24 hours and recorded accordingly. To differentiate the motile
bacteria from the non-motile ones, the motility test was done followed by the
338 Reza et al.

method mentioned by Cowan (1985) where observing the movement of bacteria,

motility were identified.
Gram staining was conducted followed by the method described by
Petersen et al. (2016) using a compound microscope.
Biochemical identification of E. coli isolates: For the verification of E. coli,
the biochemical tests performed were Voge’s Proskauer (VP) test, methyl red
(MR) test, Catalase test, TSI test, Oxidase test, urease activity test, lactose
fermentation tests, indole test, and Citrate utilization test. E. coli were
distinguished by their ability to ferment lactose, positive for indole test and MR
test and negative for VP, urease activity, Oxidase test, and Citrate utilization
test.
Preservation of bacterial isolates in pure culture form: To use the prepared
pure culture as a stock culture, it was stored at -20ºC after adding equal volume
of 80% glycerin.
Detection of Antimicrobial Sensitivity Patterns: Kirby-Bauer method of
antibiotic susceptibility testing (AST) was used to study E. coli which is known
as the disk diffusion method as well. In this experiment, we used the discs
produced by Himedia Laboratories Pvt. Limited, Mumbai. The procedure of the
antimicrobial sensitivity was used for 15 isolated E. coli.
Susceptibility test of identified E. coli and ATCC was done on Mueller Hinton
Agar and 8 different antibiotic discs such as Streptomycin, Erythromycin,
Gentamycin, Chloramphenicol, Ciprofloxacin, Co-trimethxazole, Azithromycin,
and Novobiocin were used. After preparing the media with proper manufacture’s
instruction, it was sterilized by autoclaving. Sterilized Petri dishes containing
media were allowed to solidify. The fresh nutrient broth was prepared and a
volume of 6-8ml was dispensed in each test tube which then sterilized by
autoclaving. Identified E. coli and ATCC strains were then inoculated with a
sterilized loop from pure culture and were incubated for 24 hours at 37°C.OD600
was measured by using a spectrophotometer for keeping the same bacterial
density and the OD600 of broth cultures were adjusted at 0.24 by serial dilution
method. By using L shaped glass rod, 100 microliters of microbial inoculums
from the diluted nutrient broth was poured and spread throughout the surface
of Mueller Hinton Agar. The antibiotic disc was set on the surface of the ager,
with the help of a sterilized needle at a maximum distance for getting distinct
zones. The Petri dishes were then subjected for incubation at 37 °C for 12-18
hours. In this method ATCC strain (25922) was used as a reference. By analysis
with the standard table for the zone of inhibition, all the results were detected.
The Petri dishes were examined after 12-18 hours and then calculated the zone
Surveillance of Escherichia coli 339

of complete inhibition (mm in diameter). From the analyzed data, we can get the
% of resistance, intermediate, and sensitivity pattern of the identified E. coli.

(a) (b)
Fig.1: Identification of E. coli: (a) E. coli culture on Mac Conkey’s agar plate; b) E. coli culture on EMB
agar plate.

Fig. 2: Gram's staining of bacterial isolates showing Gram-negative , small rod-shaped, pink
coloredand single or paired organisms

RESULTS AND DISCUSSION

Isolation and identification of E. coli: A total number of 12 fish samples were
collected from the different markets of Sylhet city and then screened for the
confirmation of E. coli (Fig. 1). All the examined Tilapia and Mrigal samples were
found positive with E. coli. A total 21 of E. coil isolates were isolated from 12
experimental fish samples. On the basis of samples examined, the incidence of
E. coli varies.
Motility test: Twenty-one isolates were found motile in the motility test.
340 Reza et al.

Gram’s stain: In Gram’s staining, the organism showed gram-negative,

small rod-shaped, pink color, and single or paired arrangement characteristics
(Fig. 2) under the microscope.
Biochemical tests: For the suspected bacteria, several biochemical tests
were conducted for characterization. All biochemical test results are given in
Table 1.
Table 1. Biochemical test for E. coli identification

Sample code M G MR VP C TSI O U LF IT CU

E-1 + - + - + + - - + + -
E-2 + - + - + + - - + + -
E-3 + - + - + + - - + + -
E-4 + - + - + + - - + + -
E-5 + - + - + + - - + + -
E-6 + - + - + + - - + + -
E-7 + - + - + + - - + + -
E-8 + - + - + + - - + + -
E-9 - + - - + - + - + - -
E-10 + - + - + + - - + + -
E-11 + - + - + + - - + + -
E-12 + - + - + + - - + + -
E-13 + - + - + + - - + + -
E-14 + - + - + + - - + + -
E-15 + - + - + + - - + + -
E-16 + - + - + + - - + + -
E-17 - - + + - + - + - + +
E-18 + - + - + + - - + + -
E-19 + + - + - + + - - - +
E-20 - - + + - - + + - - +
E-21 - + - - + - - + - + +

M= Motility, G= Gram test, MR= Methyl Red test, VP= Voge’s-Proskauer, C= Catalase test, TSI= Triple
Sugar Iron, O= Oxidase test, U= Urease Activity, LF= Lactose Fermentation Test, IT= Indole Test,
CU= citrate agar test.

Presumptive result: To analysis, all E. coli isolates, it was confirmed that E1,
E2, E3, E4, E5, E6, E7, E8, E10, E11, E12, E13, E14, E15, E16, and E18
isolates were E. coli. E. coli was observed as Oxidase negative, Catalase positive,
Gram-negative, Indole test positive, MR positive, VP negative, and TSI (Triple
Sugar Iodine Agar) test positive. List of presumptive E. coli isolates was identified
according to Bergey’s (1994) and Cowan & Steel (1974).
Determination of antibiotic sensitivity pattern: 16 isolates were screened for
drug resistance profile. All the isolates showed the highest degree of sensitivity
against the commonly used antibiotics (Table 2). E. coli ATCC strain was found
Surveillance of Escherichia coli 341

to be highly sensitive against the used antibiotics. Table 3 showed the Standard
clear zone diameters for Enterobacteriaceae and table 4 showed the result of this
research. The result investigate that sensitivity pattern of E. coli for 8 antibiotics
{Chloramphenicol (CH); Streptomycin (S); Gentamycin (G); Ciprofloxacin (CI);
Co-trimethoxazole (CO); Azithromycin (AZI); Erythromycin (E); Novobiocin (NV)}
were respectively 100%, 25%, 100%, 75%, 87.5%, 81.25%, 0%,0%. Whereas
resistance patterns of E. coli for 8 antibiotics were 81.25% and 87.5% for
Erythromycin (E) and Novobiocin (NV) respectably. The resistance pattern of E.
coli for the rest of the 6 antibiotics was 0% to all. On the other hand,
intermediate pattern of E. coli for 8 {Chloramphenicol (CH); Streptomycin (S);
Gentamycin (G); Ciprofloxacin (CI); Co-trimethoxazole (CO); Azithromycin (AZI);
Erythromycin (E); Novobiocin (NV) } antibiotics were respectively 0%, 75%, 0%,
25%, 12.5%, 18.75%, 18.75%, 12.5%.

Table 2. Antibiotic sensitivity and resistance pattern of isolated E. coli by measuring zone
diameter of inhibition

Sample code S E G CH CI CO AZI NV

E-1 15mm 15 23mm 25mm 21mm 26mm 18mm NZ

E-2 20mm 6mm 20mm 22mm 24mm 20mm 20mm 6mm
E-3 18mm NZ 22mm 25mm 20mm 22mm 19mm 12mm
E-4 20mm 14mm 24mm 20mm 28mm 18mm 22mm NZ
E-5 19mm 10mm 21mm 27mm 22mm 14mm 16mm NZ
E-6 22mm NZ 20mm 22mm 25mm 24mm 18mm 8mm
E-7 20mm 16mm 21mm 28mm 28mm 18mm 20mm NZ
E-8 20mm NZ 22mm 22mm 24mm 28mm 18mm 10mm
E-10 22mm NZ 24mm 23mm 20mm 20mm 20mm NZ
E-11 18mm 6mm 20mm 26mm 23mm 16mm 18mm 13mm
E-12 17mm NZ 23mm 26mm 20mm 20mm 21mm NZ
E-13 18mm 20mm 24mm 26mm 15mm 17mm NZ
E-14 22mm NZ 21mm 20mm 22mm 20mm 21mm 7mm
E-15 14mm 8mm 20mm 23mm 20mm 16mm 16mm 7mm
E-16 20mm 9mm 22mm 20mm 23mm 22mm 20mm NZ
E-18 21mm NZ 24mm 25mm 21mm 18mm 18mm 9mm

Note: S = Streptomycin; E = Erythromycin; G = Gentamycin; CH = Chloramphenicol; CI =

Ciprofloxacin; CO = Co-trimethoxazole; AZI = Azithromycin; NV = Novobiocin

The study was conducted to investigate the prevalence of Escherichia coli in

freshwater fish particularly in Tilapia (Oreochromis niloticus) and Mrigal
(Cirrhinus mrigala) in Sylhet, Bangladesh.
In this study, with the nutrient broth turbid, black centered colony with
metallic sheen was observed in EMB agar. The present results similar to the
findings of the Zinnah et al. (2007) and Sarba et al. (2019) presumptively
recognized E. coli where they found in an EMB agar a greenish-black colony
along with a metallic sheen. Same results were also found by some other
342 Reza et al.

authors (Buxton and Fraser, 1977; Freeman, 1985; Jones, 1987) whereas
characteristic features of E. coli such as gram-negative, pink color, small rod-
shaped were noticed in gram's staining as well as hanging drop technique
(Cowan, 1985) in which all the isolates showed motility. At the same time, the
study possess the similar findings of Zinnah et al. (2007), Sarba et al. (2019),
and Buxton and Fraser (1977) where the yellow slant revealed from the TSI agar
slant with no hydrogen sulphide production. E. coli was confirmed by several
biochemical tests were according to the Ali et al., (1998).

Table 3. Standard clear zone diameters for Enterobacteriaceae

Name of the antibiotics Zone diameter (mm)

Resistant Intermediate Sensitive
Azithtromycin (AZI) ≤13 14-17 ≥18 -
Ciprofloxacin (CI) ≤15 16-20 ≥21
Gentamycin (G) ≤12 13-14 ≥15
Erythromycin (E) ≤13 14-22 ≥23
Co-Trimoxazale (Sulpha/ ≤10 11-15 ≥16
Trimethoprim) (CO)
Streptomycin (S) ≤13 14-20 ≥21
Chloramphenicol (CH) ≤12 13-17 ≥18
Novobiocin (NV) ≤11 12-15 ≥16

Table 4 Antimicrobial susceptibility pattern of E. coli isolated

Name of Antibiotic Sensitivity pattern of Sensitivity pattern of E.

Antibiotics conc. isolated E. coli strains coli ATCC strain
(µg/disc) %R %I %S %R %I %S
Streptomycin 10 - 75 25 - - 100
Erythromycin 15 81.25 18.75 - 100 - -
Gentamycin 10 - - 100 - - 100
Chloramphenicol 30 - - 100 - - 100
Ciprofloxacin 5 - 25 75 - - 100
Co-trimethxazole 25 - 12.5 87.5 - - 100
Azithromycin 30 - 18.75 81.25 - - 100
Novobiocin 30 87.5 12.5 - 100 - -

These days, antimicrobial resistance became a worldwide matter of concern

(Islam et al. 2016 and Miles et al. 2006). In country like Bangladesh, the issue of
multi-drug resistant strain of E. coli is constantly raised due to the abuse of
antibiotics (Islam et al., 2016). Although this finding was found to be similar
with the many past studies, the incident was increased in food as well as human
beings by several factors such as use of non-selective antibiotics with
insufficient knowledge and at the same time carelessness towards the disease.
In the present study, the prevalence, pattern of antibiotic sensitivity of E. coli in
fish were examined.
Surveillance of Escherichia coli 343

Spreading of the drug-resistant bacteria in the sewage and surface water

occurred due to the release of human, animal, and bird fecal. Under some
defined physicochemical and biological conditions, the resistant E. coli strains
interchange R-plasmids to susceptible strains of E. coli (Rahman, 2008). When
the environment get contaminated with the drug-resistant E. coli, human as well
as animals face difficulties in the treatment after getting the infection (Joseph et
al., 1979).
Multiple-drug (Erythromycin and Novobiocin) resistance was seen in all the
strains and there is a common resistance pattern for antibiotics used among the
strains. The mechanism for the spread of antibiotic resistance must be
considered seriously like this present study. It is an alarming sign that the
percentages of sensitivity towards all the randomly selected six antibiotics
werenot satisfactory. From all of these antibiotics, Erythromycin (E) and
Novobiocin (NV) were found unable to inhibit bacterial growth so that we cannot
use them for the treatment of various diseases caused by E. coli from freshwater
fish. At present, the possible reason of this high incidence of multidrug
resistance is the non-selective use of antibiotics. With time, this might be taking
the place of drug-sensitive organisms from the antibiotic saturated
environment(Jawetz et al., 1984).
Resistance to Erythromycin is becoming a serious clinical problem (Mycek et
al., 2000). In this study, E .coli was found the height resistance to the
Erythromycin (81.25%) while Al-Ghamdi et al., (2001) found E. coli isolates from
layer and broiler source resistance to Erythromycin (87.4%).
E. coli is a common pathogen isolated from fish meal and fish farming water
(Oliveira et al. 2017 and Ristoriet al.2007), and play an important role in
economic losses among the fish industry. As E. coli is highly pathogenic and
cause diarrhoea (Sousa, 2006), so special care and further investigation are
required to tackle this situation. Moreover, it is well evidence that E. coli causes
food poisoning and food spoilage (Ekici et al. 2019). Therefore, the present study
was performed to isolate and confirmed the presence of E. coli in freshwater fish
by using various methods. From this study, it appears that E. coli collected from
freshwater fish samples have some differences in antibiotic sensitivity patterns.
Therefore, further study should include a wide variety of molecular tests with a
large number of freshwater fish samples to see the difference and variation
between them to reach a conclusive finding.

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(Manuscript received on 27 May, 2020 revised on 27 August, 2020)