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Journal of Fisheries and Aquaculture Research JFAR

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

Research Article

Isolation and Identification of Pathogenic Bacteria in Pond

Water and Fish Body and Evaluation of their Antibiotic
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,
2Department of Health Science, Edna Adan University, Hargeisa, Somalia
3Department of Fisheries Management, Hajee Mohammad Danesh Science and Technology University, Dinajpur 5200,

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

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


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:
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 MATERIALS AND METHODS

bacterial species are mostly found naturally living in the
fish habitat for example Vibrio and Aeromonas species Collection of bacterial samples
(Rodricks, 1991). The pathogenic nature of bacteria from
fish was identified when fish are physiologically unstable, Water and fish samples were collected from three (3)
nutritionally deficient, or many others factors such as, different earthen ponds of Dinajpur district of Bangladesh.
polluted water, over population in the pond i.e. The collected samples were kept in a sterile zip lock bag
overstocking, which allow possible bacterial infections to and were directly brought into the laboratory for further
prevail (Sichewo et al., 2014; Austin, 2011). Some bacteriological investigations.
pathogenic and semi-pathogenic bacteria linked with fish
and shellfish include Mycobacteium, Streptococcus spp., Determination of total bacterial count
Vibrio spp., Aeromonas spp., Salmonella spp. and others
(Sichewo et al., 2014; Lipp and Ross, 1997). Animal The total viable count was done following the procedure
manure has long been practiced in fish farming which is described by Kamal et al. (2019). Shortly, 1 mL (for water
shed directly into a fish pond as fertilizer and supports the sample) and/ 1 cm2 (for fish body) of each sample was
growth of photosynthetic organisms and contributes to the transferred to a sterilized bottle to which 9 mL sterile
growth of natural planktonic food. These plankton are used deionized water was added and mixed properly. From this
as feed for fish and also plays many other important roles solution, 1 mL of 10-1 –10-10 dilutions were made on plates
in pond aquaculture including –increasing of dissolved holding plate count agar (PCA) and incubated at 37oC for
oxygen level, which is an important elements for fish 24 h. Plates containing 30-300 colonies were used for
growth and the most important sink of ammonia-nitrogen, counting and overloaded plates were discarded. Colonies
which is evacuated by the fish species (Teichert- were counted using a haemacytometer (Labtronics, Model
Coddington and Green, 1993). However, the most matter No. 37, Korea). Finally, the total bacterial presence in the
of concern that the use of various livestock fertilizer/feed sample was calculated using the following equation:
in fish culture resulted in the increase of pathogenic
bacteria that can causes a serious public health hazards Total viable count (CFU/mL or CFU/cm2 )
(Musaiger and Souza, 2008). It is reported that Number of colony × reciprocal of dilution
consumption of contaminated fish on can be an important =
Sample taken (mL or cm2 )
route for the entry of pathogenic bacteria in human body,
which may cause food borne diseases such as, dysentery, Isolation of bacterial species
typhoid, fever, salmonellosis and cholera. to man
(Sichewo et al., 2014; Christopher et al., 2014). Therefore, Bacterial species were isolated from the collected samples
fish farming needs to be improved in a perspective way using different culturing techniques. For these purpose,
considering the possible health risks (FAO, 2003). different culturing medium such as nutrient agar,
Previous works reported that, a number of livestock MacConkey, Eosin Methylene Blue, Salmonella Shigella
manure are contaminated with pathogenic bacteria such (SS), Mannitol Salt and Cetrimide agar were prepared
as Salmonella, Shigella, Pseudomonas, Vibrio, according to their standard method. Thereafter, collected
Streptococcus, and E. coli species (Sichewo et al., 2014; samples were initially cultured in nutrient agar media at
Abdelhamid et al., 2006). Due to lack of proper cooking of 37oC for 24 hr. The following steps were followed to isolate
food or poor handling of fish, these pathogens are specific bacterial prevalence: (a) Circular yellowish
transmitting to the human body. It is also reported that a colonies from nutrient agar were inoculated directly into
great economic loss can happen due to food borne illness mannitol salt media (selective media for Staphylococcus)
such as dysentery and diarrhea resulting from and incubated at 37oC for 24 hr.; (b) The cultivated
consumption of contaminated fish and such can be a organisms from nutrient agar were inoculated directly into
serious problem to the immune system of children and MacConkey agar and incubated at 37oC for 24 hr.; (c)
elderly people (Sichewo et al., 2014), which is a great Lactose fermenting pink colony from MacConkey was sub
concern for public health. In addition, the number of cultured into eosin methylene blue agar, used selective
resistant strains of different microbial species is increasing media for E. coli and Klebsiella spp. and incubated at 37oC
day by day because of the use of antibiotics which can for 24 hr.; (d) The non-lactose fermenting colorless colony
influence their adaptation in these environment (Zampieri from the MacConkey agar was sub-cultured on SS agar
et al., 2017). The microbial association with fish media (selective media for pathogenic Salmonella spp.
compromises safety and the quality for human and Shigella) and incubated at 37oC for 24 hr.; and (e) The
consumption; particularly critical is when the micro- cultivated organisms from nutrient agar were inoculated
organisms are opportunistic and/ pathogenic in nature directly into the cetrimide agar media (selective media for
(Mhango et al., 2010). Considering the above points, the Pseudomonas spp.) and incubated at 37oC for 24 hr.
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
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
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 properties of Salmonella spp. another isolate (C) displayed
mucoid pink lactose fermenter colony and pinkish colony
Bacterial species were isolated based on their cultural on the MacConkey agar and Eosin methylene blue agar,
properties. For these, culture medium such as nutrient respectively, while no growth was found on the others
agar, MacConkey, Eosin Methylene Blue, Salmonella medium, which means the isolates might be Klebsiella.
Shigella (SS), Mannitol Salt and Cetrimide agar were The isolate D produce the colorless pale non lactose
used. The results obtained were summarized in Table 1. It fermenter colony and translucent colorless colony on the
can be seen that isolate A showed rose pink lactose MacConkey agar and SS agar, respectively, however, no
fermenter colony on the MacConkey agar medium and growth occurred on the other mediums, which might be the
moist circular colonies with dark centers yellow green properties of Shigella. The isolate E exhibited the smooth
metallic sheen on Eosin methylene blue agar, however, no non lactose fermenting colony and short rod type colony
growth was occurred in Salmonella-Shigella (SS), on the MacConkey agar and cetrimide agar medium,
Mannitol Salt and Cetrimide agar, which suspect the respectively while no growth on other medium, which
isolated species as E. coli. Isolate B exhibited Colorless showed the properties of Pseudomonas. The final isolate
pale translucent colony and translucent colorless smooth F showed the yellowish colony on the mannitol salt agar
colony with dark black centers on the MacConkey agar and while no growth on others medium, therefore, this isolate
SS agar, respectively, however, no growth occurred on the may be Staphylococcus.
other mediums. These characteristics showed the

Table 1. Morphological characterization of bacterial pathogens from pond water and fish body surface by cultural
Bacterial Salmonella- Mannitol Cetrimide
MacConkey agar Eosin Methylene blue bacterial
Isolates Shigella agar salt agar agar
Moist circular colonies
Rose pink lactose
A with dark centers yellow No growth No growth No growth E. coli
fermenter colony
green metallic sheen
Translucent colorless
Colorless pale
B No growth smooth colony with No growth No growth Salmonella spp.
dark black centers
Mucoid pink lactose
C Pinkish colony No growth No growth No growth Klebsiella
fermenter colony
Colorless pale non
Translucent colorless
D lactose fermenter No growth No growth No growth Shigella
Smooth non lactose
E No growth No growth No growth Short rod Pseudomonas
fermenting colony
F No growth No growth No growth No growth Staphylococcus

Identification and characterization Identification by biochemical tests

Identification by Gram’s staining technique For more confirmation of their presence i.e. identification
Primarily isolated bacterial species were identified by the of these bacterial species different biochemical tests were
Gram’s staining method, where different bacteria were carried out and the results obtained were presented in
differentiated using their color, shape, arrangement and Table 3. In this study, E. coli, Salmonella, Klebsiella,
gram’s responses (Table 2). Isolates A, B, C and D were Shigella, Pseudomonas, and Staphylococcus were
pink colored, short plumps rod shaped with single paired identified from the fish water and fish body based on the
or short chain arrangement which negatively response to biochemical tests. The fish are highly fragile and prone to
the Gram’s staining, therefore, identified as E. coli, quick variations in quality because of species,
Salmonella spp, Shigella and Klebsiella, respectively. On environmental conditions, and feeding habits (Rani et al.,
the otherhand, isolates E was fluorescent colored, rod 2016). They are able to create several microbial and other
shaped with bacillus arrangement which negatively health hazards. Although only a few infectious agents in
response to the Gram’s staining, therefore, identified as fish are able to infect humans, some exceptions exist that
Pseudomonas species. The last isolate F was violet may result in fatalities, which possess the risk to human
colored, cocci like shape with grape like cluster, which health due to the consumption of raw or insufficiently
positively response to the Gram’s staining and identified processed fish and fish products. Therefore, maintenance
as Staphylococcus. Therefore, most of the isolated of quality is the most important things in production and
bacterial species were gram negative. trade of fishes and fishery products.

Isolation and Identification of Pathogenic Bacteria in Pond Water and Fish Body and Evaluation of their Antibiotic Susceptibility
J. Fish. Aquacul. Res. 058

Table 2. Characterization of isolated bacteria pathogens from pond water and fish body surface by Gram’s staining
Color Shape Arrangement Grams staining Identified as
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,

ciprofloxacin, and tetracycline, the resilient properties
The isolated bacterial pathogens were evaluated for their against amoxicillin and erythromycin while intermediary
ability to resist against selected antibiotics and the results sensitive to chloramphenicol. From Table 4, it was found
obtained are presented in Table 4. It can be seen that E. that most of the isolated pathogens become unaffected by
coli was most sensitive to gentamycin and ciprofloxacin the studied antibiotics while some antibiotics showed their
while intermediary sensitive to tetracycline. On the other sensitivity to some of the pathogens isolated from the fish
hand, amoxicillin, erythromycin and chloramphenicol pond and fish body surface. Previous studies evidenced
showed resistant by the E. coli. Salmonella showed its that use of different manure in fish culture are associated
sensitivity to gentamycin, ciprofloxacin and amoxicillin with the antibiotics resistance properties of pathogenic
while resistance to erythromycin and tetracycline. bacteria. This may be because of the use of some low
Klebsiella showed resistance against the antibiotics levels of antibiotics directly on the fish ponds and/or farms
amoxicillin and erythromycin, and intermediate sensitive to for the treatment and/or growth promotion (Wamala et al.,
gentamycin. However, sensitivity properties were found 2018). Furthermore, transfer of resistant strains of bacteria
against ciprofloxacin, tetracycline and chloramphenicol. from humans and livestock to fish body (Van den Bogaard
Another pathogen Shigella exhibited its sensitivity and Stobberingh 2000; Akinbowale et al., 2006) may also
properties against gentamycin and tetracycline while contributed to the fact. In addition, use of animal wastes to
intermediary sensitive to ciprofloxacin. However, fertilize fish ponds (Sørum and Sunde, 2001; Shah et al.,
resistance property was found when treated against 2012) and the naturally occurring resistant bacterial strains
amoxicillin, erythromycin and chloramphenicol. in the aquatic environment and soils (Cantas et al. 2013)
Pseudomonas, one of the most important spoilage and could contribute directly to this effect by passing over
pathogenic bacteria in foodstuffs showed sensitivity antibiotic resistance genes to fish bacteria. There is a high
against most of the antibiotics studied including risk of infection and spread of bacteria when animals are
gentamycin, amoxicillin, tetracycline and chloramphenicol living in high densities like in intensive aquaculture
while resistant to ciprofloxacin and erythromycin. (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 FAO (2003). Recycling of animal wastes as a source of

nutrients for freshwater fish culture within an integrated
The present study was conducted in order to isolate, livestock system.
identify and characterize the bacteria from fish and pond C526E/AC526E01.htm
water from different farms in Dinajpur district. The study Kamal MM, Rashid MH, Mondal SC, El Taj HF, Jung C
shown that the predominance of bacterial pathogens (2019). Physicochemical and microbiological
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coli 12.5%, Salmonella spp. 31.25%, Klebsiella spp feeding of bees. Journal of Food Science and
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Isolation and Identification of Pathogenic Bacteria in Pond Water and Fish Body and Evaluation of their Antibiotic Susceptibility