Journal of Fisheries and Aquaculture Research JFAR

Vol. 5(1), pp. 054-060, March, 2020. © www.premierpublishers.org, ISSN: 9901-8810

Research Article

Isolation and Identification of Pathogenic Bacteria in Pond

Water and Fish Body and Evaluation of their Antibiotic
Susceptibility
Mohamed Abdikadir Jama1,2*, Omar Hassan Mohammed3, Abdirizak Ahmed Guled4, Md. Khaled
Hossain5, Mir Rowshan Akter6
1,5,6Department of Microbiology, Hajee Mohammad Danesh Science and Technology University, Dinajpur 5200,
Bangladesh
2Department of Health Science, Edna Adan University, Hargeisa, Somalia
3Department of Fisheries Management, Hajee Mohammad Danesh Science and Technology University, Dinajpur 5200,

Bangladesh
4Department of Food Processing and Preservation, Hajee Mohammad Danesh Science and Technology University,

Dinajpur 5200, Bangladesh

This research was focused on the isolation and identification of pathogenic bacterial species in
pond water and fish body of selected fish farms(ponds) of Bangladesh. Also, resistance to
antibiotics such as gentamycin, ciprofloxacin, amoxicillin, erythromycin, tetracycline and
chloramphenicol was evaluated. For this, the collected samples were subjected to different
bacteriological tests for isolation and identification of bacteria based on morphology, staining,
cultural and biochemical, and also antibiotic sensitivity examinations. Total viable count was
obtained as 1.41×107 – 3.57×107 CFU/cm2 in the fish body and 1.84×107 -4.40×107 CFU/cm2 in the
fish pond. The study shown that among the isolated species of bacteria, the most prevalence in
pond water was Salmonella spp. (18.75-31.25%) followed by Shigella spp. (6.25-18.75%),
Staphylococcus spp. (6.25-18.75%), Pseudomonas spp. (6.25-12.5%), E. coli (6.25-12.5%) and
Klebsiella spp. (6.25%). while the highest frequency in fish body was observed as Salmonella spp.
(31.25-43.75%), Shigella spp. (12.5-18.75%), E. coli (12.5-18.75%), Klebsiella spp. (6.25%),
Staphylococcus spp. (6.25%) and Pseudomonas spp. (6.25%). The isolated bacterial species
showed resistance properties against some antibiotics however, gentamycin, ciprofloxacin,
erythromycin and kanamycin were identified as effective against these bacterial species. This
research exposed that the studied pond water and fish samples were of serious concern due its
harborness of different bacterial species, which have potential to cause health hazards to the
consumers.

Keywords: Fish, pond, bacteria, isolation, identification, antibiotics

INTRODUCTION

The microbiological diversity of fresh fish species depends coli, Clostridium botulinum, Shigella dysenteriae,
on the fishing practices and environmental conditions Staphylococcus aureus, Listeria monocytogens and
around it (Cahill, 1990) and the type of microorganisms Salmonella, which contaminate the fishing environment
associated with particular fish depends on its habitat
(Sichewo et al., 2014; Claucas and Ward, 1996). The *Corresponding Author: Mohamed Abdikadir Jama;
bacterial pathogens associated with fish have been Department of Microbiology, Hajee Mohammad Danesh
classified as indigenous and non-indigenous (Kvenberg, Science and Technology University, Dinajpur 5200,
1991). Some non-indigenous species include Escherichia Bangladesh. Email: maj.hstu@gmail.com
Isolation and Identification of Pathogenic Bacteria in Pond Water and Fish Body and Evaluation of their Antibiotic Susceptibility
Jama et al. 055
(Sichewo et al., 2014). The indigenous pathogenic
bacterial species are mostly found naturally living in the
fish habitat for example Vibrio and Aeromonas species
(Rodricks, 1991). The pathogenic nature of bacteria from
fish was identified when fish are physiologically unstable,
nutritionally deficient, or many others factors such as,
polluted water, over population in the pond i.e.
overstocking, which allow possible bacterial infections to
prevail (Sichewo et al., 2014; Austin, 2011). Some
pathogenic and semi-pathogenic bacteria linked with fish
and shellfish include Mycobacteium, Streptococcus spp.,
Vibrio spp., Aeromonas spp., Salmonella spp. and others
(Sichewo et al., 2014; Lipp and Ross, 1997). Animal
manure has long been practiced in fish farming which is
shed directly into a fish pond as fertilizer and supports the
growth of photosynthetic organisms and contributes to the
growth of natural planktonic food. These plankton are used
as feed for fish and also plays many other important roles
in pond aquaculture including –increasing of dissolved
oxygen level, which is an important elements for fish
growth and the most important sink of ammonia-nitrogen,
which is evacuated by the fish species (Teichert-
Coddington and Green, 1993). However, the most matter
of concern that the use of various livestock fertilizer/feed
in fish culture resulted in the increase of pathogenic
bacteria that can causes a serious public health hazards
(Musaiger and Souza, 2008). It is reported that
consumption of contaminated fish on can be an important
route for the entry of pathogenic bacteria in human body,
which may cause food borne diseases such as, dysentery,
typhoid, fever, salmonellosis and cholera. to man
(Sichewo et al., 2014; Christopher et al., 2014). Therefore,
fish farming needs to be improved in a perspective way
considering the possible health risks (FAO, 2003).
Previous works reported that, a number of livestock
manure are contaminated with pathogenic bacteria such
as Salmonella, Shigella, Pseudomonas, Vibrio,
Streptococcus, and E. coli species (Sichewo et al., 2014;
Abdelhamid et al., 2006). Due to lack of proper cooking of
food or poor handling of fish, these pathogens are
transmitting to the human body. It is also reported that a
great economic loss can happen due to food borne illness
such as dysentery and diarrhea resulting from
consumption of contaminated fish and such can be a
serious problem to the immune system of children and
elderly people (Sichewo et al., 2014), which is a great
concern for public health. In addition, the number of
resistant strains of different microbial species is increasing
day by day because of the use of antibiotics which can
influence their adaptation in these environment (Zampieri
et al., 2017). The microbial association with fish
compromises safety and the quality for human
consumption; particularly critical is when the micro-
organisms are opportunistic and/ pathogenic in nature
(Mhango et al., 2010). Considering the above points, the
current study was aimed to isolate and identify different
pathogenic microorganisms in water and fish body and
also to evaluate the resistance capacity of identified
pathogens to selected antibiotics.
Isolation and Identification of Pathogenic Bacteria in Pond Water and Fish Body and Evaluation of their Antibiotic Susceptibility
MATERIALS AND METHODS
Collection of bacterial samples
Water and fish samples were collected from three (3)
different earthen ponds of Dinajpur district of Bangladesh.
The collected samples were kept in a sterile zip lock bag
and were directly brought into the laboratory for further
bacteriological investigations.
Determination of total bacterial count
The total viable count was done following the procedure
described by Kamal et al. (2019). Shortly, 1 mL (for water
sample) and/ 1 cm2 (for fish body) of each sample was
transferred to a sterilized bottle to which 9 mL sterile
deionized water was added and mixed properly. From this
solution, 1 mL of 10-1 –10-10 dilutions were made on plates
holding plate count agar (PCA) and incubated at 37oC for
24 h. Plates containing 30-300 colonies were used for
counting and overloaded plates were discarded. Colonies
were counted using a haemacytometer (Labtronics, Model
No. 37, Korea). Finally, the total bacterial presence in the
sample was calculated using the following equation:
Total viable count (CFU/mL or CFU/cm2 )
Number of colony × reciprocal of dilution
=
Sample taken (mL or cm2 )
Isolation of bacterial species
Bacterial species were isolated from the collected samples
using different culturing techniques. For these purpose,
different culturing medium such as nutrient agar,
MacConkey, Eosin Methylene Blue, Salmonella Shigella
(SS), Mannitol Salt and Cetrimide agar were prepared
according to their standard method. Thereafter, collected
samples were initially cultured in nutrient agar media at
37oC for 24 hr. The following steps were followed to isolate
specific bacterial prevalence: (a) Circular yellowish
colonies from nutrient agar were inoculated directly into
mannitol salt media (selective media for Staphylococcus)
and incubated at 37oC for 24 hr.; (b) The cultivated
organisms from nutrient agar were inoculated directly into
MacConkey agar and incubated at 37oC for 24 hr.; (c)
Lactose fermenting pink colony from MacConkey was sub
cultured into eosin methylene blue agar, used selective
media for E. coli and Klebsiella spp. and incubated at 37oC
for 24 hr.; (d) The non-lactose fermenting colorless colony
from the MacConkey agar was sub-cultured on SS agar
media (selective media for pathogenic Salmonella spp.
and Shigella) and incubated at 37oC for 24 hr.; and (e) The
cultivated organisms from nutrient agar were inoculated
directly into the cetrimide agar media (selective media for
Pseudomonas spp.) and incubated at 37oC for 24 hr.
 J. Fish. Aquacul. Res. 056

Identification of isolated pathogens

Identification by Grams staining method

The representative bacterial colonies were characterized

morphologically using Gram’s stain based on the protocol
described by Merchant and Packer (1967). Briefly, a
smear of each of the organisms was prepared. The
smears were flooded with crystal violet and let stood for 1
min and washed with tap water. After that the smears were
flooded with Grams iodine mordant and let stood for 1 min
and washed with tap water. Then, the smears were
decolorized with 95% ethyl alcohol and washed with tap
water. Finally, the smears were counter stain with safranin
for 30s and washed with tap water. Then the slide was Figure 1. Total viable count of pond water and fish surface
blotted with blot paper and was allowed to dry. The slide
was examined under microscope with high power to
characterize the bacterial species based on color, shape, RESULTS AND DISCUSSION
and arrangements. Finally, Gram positive (violate colored)
and gram negative (pink colored) organisms were Prevalence of bacterial pathogens
selected.
The total viable bacterial count was showed in Figure 1. It
Identification by biochemical tests is seen that fish body (1.84×107 -4.40×107 CFU/cm2 with
an average of 2.96×107 CFU/cm2) contained higher
To confirm the identification of isolated bacterial species, bacterial pathogens than the pond water (1.41×107 –
following tests were applied: indole, methyl red (MR), 3.57×107 CFU/mL with an average of 2.72×107 CFU/mL).
Voges Proskauer, and triple sugar iron (TSI). Previous study of Njoku et al. (2015) determined the
microbial profile of some concrete and earthen fish ponds
Evaluation of Antibiotic susceptibility within the Niger delta region and reported that the mean
total heterotrophic bacteria count was higher in concrete
The antibiotic susceptibility of isolated bacterial species ponds (6.5×105 to 7.4×105 CFU/mL). The prevalence of
was evaluated using the disc diffusion method (Moore et different bacterial species existent in the samples was
al. 2014). Briefly, a cotton swab was charged with presented in Figure 2. Among the bacterial species,
inoculum of the isolated bacterial population and was Salmonella (18.75-31.25%) showed the highest
inoculated onto the surface of Mueller–Hinton agar (Oxoid prevalence followed by the Shigella (6.25-18.75%),
CM0337). On drying, a standard disk diffusion assay was Staphylococcus (6.25-18.75%), Pseudomonas (6.25-
performed, where antibiotic disks of the following antibiotic 12.50%), E. coli (6.25-12.50%) and Klebsiella (6.25%) for
agents were placed onto the surface with a semi- different ponds water samples whereas Salmonella
automated stamper: - Gentamycin, Ciprofloxacin, (31.25-43.75%) showed the highest prevalence followed
Amoxicillin, Erythromycin, Tetracycline and by the E. coli (12.50-18.75%), Shigella (12.50-18.75%),
Chloramphenicol. Plates were incubated at 20oC for 48 h Staphylococcus (6.25%), Pseudomonas (6.25%), and
prior to reading. Resistance was recorded as any bacterial Klebsiella (6.25%) for fish body of the selected ponds.
morphology growing to the edge of the antibiotic disk,
whereas sensitivity was defined as a complete clear zone
of inhibition for all bacterial taxa represented on the
susceptibility agar.

Statistical analysis

All the values mentioned in the manuscript were analyzed

using the SPSS (version 20.0) and presented as the mean
with standard deviation of three replicates.

Figure 2. Prevalence of isolated bacterial species from

pond water and fish surface

Isolation and Identification of Pathogenic Bacteria in Pond Water and Fish Body and Evaluation of their Antibiotic Susceptibility
Jama et al. 057
Isolation of bacterial pathogens
Bacterial species were isolated based on their cultural
properties. For these, culture medium such as nutrient
agar, MacConkey, Eosin Methylene Blue, Salmonella
Shigella (SS), Mannitol Salt and Cetrimide agar were
used. The results obtained were summarized in Table 1. It
can be seen that isolate A showed rose pink lactose
fermenter colony on the MacConkey agar medium and
moist circular colonies with dark centers yellow green
metallic sheen on Eosin methylene blue agar, however, no
growth was occurred in Salmonella-Shigella (SS),
Mannitol Salt and Cetrimide agar, which suspect the
isolated species as E. coli. Isolate B exhibited Colorless
pale translucent colony and translucent colorless smooth
colony with dark black centers on the MacConkey agar and
SS agar, respectively, however, no growth occurred on the
other mediums. These characteristics showed the
Table 1. Morphological characterization of bacterial pathogens from pond water and fish body surface by cultural
properties.
Bacterial Salmonella-
MacConkey agar Eosin Methylene blue
Isolates Shigella agar
Moist circular colonies
Rose pink lactose
A with dark centers yellow No growth
fermenter colony
green metallic sheen
Translucent colorless
Colorless pale
B No growth smooth colony with No growth No growth Salmonella spp.
translucent
dark black centers
Mucoid pink lactose
C Pinkish colony No growth
fermenter colony
Colorless pale non
Translucent colorless
D lactose fermenter No growth
colony
colony
Smooth non lactose
E No growth No growth
fermenting colony
F No growth No growth No growth
Identification and characterization
Identification by Gram’s staining technique
Primarily isolated bacterial species were identified by the
Gram’s staining method, where different bacteria were
differentiated using their color, shape, arrangement and
gram’s responses (Table 2). Isolates A, B, C and D were
pink colored, short plumps rod shaped with single paired
or short chain arrangement which negatively response to
the Gram’s staining, therefore, identified as E. coli,
Salmonella spp, Shigella and Klebsiella, respectively. On
the otherhand, isolates E was fluorescent colored, rod
shaped with bacillus arrangement which negatively
response to the Gram’s staining, therefore, identified as
Pseudomonas species. The last isolate F was violet
colored, cocci like shape with grape like cluster, which
positively response to the Gram’s staining and identified
as Staphylococcus. Therefore, most of the isolated
bacterial species were gram negative.
Isolation and Identification of Pathogenic Bacteria in Pond Water and Fish Body and Evaluation of their Antibiotic Susceptibility
properties of Salmonella spp. another isolate (C) displayed
mucoid pink lactose fermenter colony and pinkish colony
on the MacConkey agar and Eosin methylene blue agar,
respectively, while no growth was found on the others
medium, which means the isolates might be Klebsiella.
The isolate D produce the colorless pale non lactose
fermenter colony and translucent colorless colony on the
MacConkey agar and SS agar, respectively, however, no
growth occurred on the other mediums, which might be the
properties of Shigella. The isolate E exhibited the smooth
non lactose fermenting colony and short rod type colony
on the MacConkey agar and cetrimide agar medium,
respectively while no growth on other medium, which
showed the properties of Pseudomonas. The final isolate
F showed the yellowish colony on the mannitol salt agar
while no growth on others medium, therefore, this isolate
may be Staphylococcus.
Possible
Mannitol Cetrimide
bacterial
salt agar agar
species
No growth No growth E. coli
No growth No growth Klebsiella
No growth No growth Shigella
No growth Short rod Pseudomonas
Yellowish
No growth Staphylococcus
colony
Identification by biochemical tests
For more confirmation of their presence i.e. identification
of these bacterial species different biochemical tests were
carried out and the results obtained were presented in
Table 3. In this study, E. coli, Salmonella, Klebsiella,
Shigella, Pseudomonas, and Staphylococcus were
identified from the fish water and fish body based on the
biochemical tests. The fish are highly fragile and prone to
quick variations in quality because of species,
environmental conditions, and feeding habits (Rani et al.,
2016). They are able to create several microbial and other
health hazards. Although only a few infectious agents in
fish are able to infect humans, some exceptions exist that
may result in fatalities, which possess the risk to human
health due to the consumption of raw or insufficiently
processed fish and fish products. Therefore, maintenance
of quality is the most important things in production and
trade of fishes and fishery products.
 J. Fish. Aquacul. Res. 058

Table 2. Characterization of isolated bacteria pathogens from pond water and fish body surface by Gram’s staining
technique.
Bacterial
Color Shape Arrangement Grams staining Identified as
Isolates
A Pinkish color Short plumps rods Single paired or short chain Gram negative E. coli
B Pinkish color Very short plump rods Single paired or short chain Gram negative Salmonella spp.
C Pinkish color Small rod shaped Single paired or short chain Gram negative Klebsiella
D Pinkish color Small rod shaped Single paired or short chain Gram negative Shigella
E Fluorescent color Rod shaped bacillus Gram negative Pseudomonas
F Violet color Cocci arranged Grape like cluster Gram positive Staphylococcus

Table 3. Results of biochemical tests on isolated bacterial species from pond water and fish body surface.
Biochemical tests
Isolated bacterial pathogens
Mortility Indole MR VP TSI
E. coli P P P N Acid & gas production (+), H2S (-)
Salmonella - N P N Acid & gas production (+), H2S (-)
Klebsiella N N N N Acid & gas production (+), H2S (-)
Shigella - P P N Acid & gas production (-), H2S (-)
Pseudomonas N N N N N
Staphylococcus N N P P Acid & gas production (-), H2S (+)
MR – Methyl red; VP – Voges Proskauer; TSI – Triple Sugar Iron; P – Positive; N – Negative

Antibiotic susceptibility of isolated pathogens Staphylococcus showed sensitivity against gentamycin,

The isolated bacterial pathogens were evaluated for their
ability to resist against selected antibiotics and the results
obtained are presented in Table 4. It can be seen that E.
coli was most sensitive to gentamycin and ciprofloxacin
while intermediary sensitive to tetracycline. On the other
hand, amoxicillin, erythromycin and chloramphenicol
showed resistant by the E. coli. Salmonella showed its
sensitivity to gentamycin, ciprofloxacin and amoxicillin
while resistance to erythromycin and tetracycline.
Klebsiella showed resistance against the antibiotics
amoxicillin and erythromycin, and intermediate sensitive to
gentamycin. However, sensitivity properties were found
against ciprofloxacin, tetracycline and chloramphenicol.
Another pathogen Shigella exhibited its sensitivity
properties against gentamycin and tetracycline while
intermediary sensitive to ciprofloxacin. However,
resistance property was found when treated against
amoxicillin, erythromycin and chloramphenicol.
Pseudomonas, one of the most important spoilage and
pathogenic bacteria in foodstuffs showed sensitivity
against most of the antibiotics studied including
gentamycin, amoxicillin, tetracycline and chloramphenicol
while resistant to ciprofloxacin and erythromycin.
ciprofloxacin, and tetracycline, the resilient properties
against amoxicillin and erythromycin while intermediary
sensitive to chloramphenicol. From Table 4, it was found
that most of the isolated pathogens become unaffected by
the studied antibiotics while some antibiotics showed their
sensitivity to some of the pathogens isolated from the fish
pond and fish body surface. Previous studies evidenced
that use of different manure in fish culture are associated
with the antibiotics resistance properties of pathogenic
bacteria. This may be because of the use of some low
levels of antibiotics directly on the fish ponds and/or farms
for the treatment and/or growth promotion (Wamala et al.,
2018). Furthermore, transfer of resistant strains of bacteria
from humans and livestock to fish body (Van den Bogaard
and Stobberingh 2000; Akinbowale et al., 2006) may also
contributed to the fact. In addition, use of animal wastes to
fertilize fish ponds (Sørum and Sunde, 2001; Shah et al.,
2012) and the naturally occurring resistant bacterial strains
in the aquatic environment and soils (Cantas et al. 2013)
could contribute directly to this effect by passing over
antibiotic resistance genes to fish bacteria. There is a high
risk of infection and spread of bacteria when animals are
living in high densities like in intensive aquaculture
(Penders and Stobberingh, 2008).

Table 4. Antibiotic sensitivity of isolated bacterial species from pond water and fish body surface.
Isolated bacterial Antibiotics tested
pathogens Gentamycin Ciprofloxacin Amoxicillin Erythromycin Tetracycline Chloramphenicol
E. coli S S R R I R
Salmonella S S S R R S
Klebsiella I S R R S S
Shigella S I R R S R
Pseudomonas S R S R S S
Staphylococcus S S R R S I
I – Intermediate; R – Resistant; S - Sensitive

Isolation and Identification of Pathogenic Bacteria in Pond Water and Fish Body and Evaluation of their Antibiotic Susceptibility
Jama et al. 059
CONCLUSION
The present study was conducted in order to isolate,
identify and characterize the bacteria from fish and pond
water from different farms in Dinajpur district. The study
shown that the predominance of bacterial pathogens
isolated and identified from pond water samples were E.
coli 12.5%, Salmonella spp. 31.25%, Klebsiella spp
6.25%, Shigella spp. 18.75%, Staphylococcus spp.
18.75% and Pseudomonas spp 12.5%. The bacterial
isolated from fish body surface were E. coli 18.75%,
Salmonella spp. 43.75%, Klebsiella spp. 6.25%, Shigella
spp. 18.75%, Staphylococcus spp. 6.25% and
Pseudomonas spp. 6.25%. Most effective antibiotics
against Salmonella spp., Shigella spp. Pseudomonas
spp., Staphylococcus spp. were Gentamycin,
Ciprofloxacin, Erythromycin and Kanamycin. In the context
of this study, it may be concluded that the presence of E.
coli, Salmonella spp. Klebsiella spp., Shigella spp.,
Staphylococcus and pseudomonas presents health
hazards to both consumers and fish farm workers such as
typhoid, dysentery, diarrhea, cholera and gastroenteritis.
The antibiotic resistance properties of isolated bacteria
can cause serious health hazards because of ineffective
treatment of the suffers by the commonly described
antibiotics.
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Isolation and Identification of Pathogenic Bacteria in Pond Water and Fish Body and Evaluation of their Antibiotic Susceptibility