The International Journal of Computational Science, Information Technology and Control Engineering (IJCSITCE)

The International Journal of Computational Science, Information Technology and Control Engineering
(IJCSITCE) Vol.10, No.4, October 2023
DOI: 10.5121/ijcsitce.2023.10401 1
PREVALENCE OF SALMONELLA ENTERICA AND
SHIGELLA FLEXNERI IN FRESH FISH PRODUCTS
SOLD AT INFORMAL MARKET ALONG THE
SHORELINE OF SOME LAKES IN ETHIOPIA
Guta Dissasa and Brook Lemma
1
Institute of Biotechnology, Addis Ababa University, Addis Ababa, Ethiopia,
2
Department of Zoological Sciences, College of Natural and Computational Sciences,
Addis Ababa University, Addis Ababa, Ethiopia
ABSTRACT
Food borne diseases are considered a major public health challenge worldwide due to their incidence,
associated mortality, and negative economic repercussions. Fish and fish products are one of the most
important sources of food borne disease and well known medium favoring growth of several bacterial
pathogens. This cross-sectional study was conducted to assess the occurrence and prevalence of
Salmonella enterica and Shigella flexneri in fresh fish products of two common fish species - Nile tilapia
(Oreochromis niloticus) and common carp (Cyprinus carpio) sold along the shoreline of two Ethiopian
rift-valley Lakes Hawassa and Ziway. Among the examined 60 samples collected from the study areas,
33(55%) were positive for S. enterica and S. flexneri. This has implications for the consumer health and
adherence to simple hygienic steps is advisable. Further, establishing a practice of regular inspection of
fish products and fish processing channels for bacterial pathogens and environmental sanitation is
necessary.
KEYWORDS
Fresh fish products; common carp (Cyprinus carpio), Nile tilapias (Oreochromis niloticus), Salmonella
enterica, Shigella flexneri
1. INTRODUCTION
Food borne diseases have been an issue for all societies since the beginning of humanity. Types,
severity and impacts of these illnesses have changed through the ages and are still diverse across
regions, countries and communities [1]. Only fractions of the people who become sick from food
they have eaten seek medical care and reported to public health authorities, and recorded in
official disease statistics. Certain chronic diseases, such as cancer, kidney or liver failure, that
result from contaminated food appear long after the ingestion of food and the causal link is never
made for each case[1, 2].
Microorganisms play a crucial and unique role in fish and fish products safety. The presences of
human bacterial pathogens make the control of pathogenic microorganisms critical for fish
products safety [2, 3]. Fish plays an important role in human diet; the production and
consumption of fish and fish products have increased worldwide over the years. However, fish
and fish products have been involved in various food borne outbreaks [3].Fish and fish products
are one of important sources of food borne disease and they are well known medium that favors

2
growth of several bacterial pathogens such as S. enterica and S. flexneri. Salmonella enteric are
responsible for non-typhoidal infection and the second leading cause of food borne illness which
can result in the patient’s death [2].
Foodstuff contamination may occur directly from infected food-producing animals or may result
from poor hygiene during production processes, or the retail and storage of food, since may also
harbor microorganisms [2, 4].Non-typhoidal Salmonella enterica spp. infection from
contaminated food is an important cause of both sporadic gastroenteritis and outbreaks
internationally. A wide variety of foods can be contaminated with Shigella including fish
products. Shigella flexneri is the most prevalent species of Shigella associate with illness in
developing countries [5]. Salmonella enterica and S. flexneri are an important cause of food
borne illness, regularly causing high-profile outbreaks involving commercially-available foods
[4].
Salmonella is a leading cause of bacterial food borne illness in the world [6]. Although S.
enterica and S. flexneri are typically associated with foods of animal origin including Milk, Eggs
and egg-containing foods, aquatic foods, such as fish and fish products, are quickly gaining
recognition as an important vehicle of Salmonella and Shigella exposure. Reports on food borne
bacterial pathogens in fish products particularly S. enterica and S. flexneri are scant in Ethiopia.
Hence, this study was aimed to assess the occurrence and prevalence of S. enterica and S. flexneri
in fish products of two common fish species - Nile tilapia (Oreochromis niloticus) and common
carp (Cyprinus carpio) sold at the informal market along the shoreline of two Ethiopian rift-
valley Lakes Hawassa and Ziway.
2. METHODS
2.1. The Study Area
The study was on two Ethiopian rift-valley lakes, Lake Ziway and Lake Hawassa, which are
well-known for their common fish catches and tourist destinations. These lakes are some 163and
275 km to the south Addis Ababa respectively (Fig. 1). The information sources were from
World Lake Database [7].
Fig. 1: The study area: (Lake Hawassa and Lake Ziway)

3
2.2. Study design and sampling strategy
The study design was cross-sectional and its period was from June to December 2022. For
sampling, the two lakes were selected purposively that the lakes are known for their common fish
catches from which fish are continuously harvested for subsistence and fresh fish products were
sold informally along their shoreline for cash [8]. Fresh fish products samples caught from the
two lakes was collected from the informal Market around the shorelines of each lake. They were
packed in a sterile container with ice blocks to prevent sample deterioration on transit [9, 10] and
transferred to the laboratory for identification and biological assays.
2.3. Sample collection
A total of 60(30 from each lake with 30 fresh Nile tilapias fish and 30 fresh common carps fish)
samples of fresh fish products those caught from Lake Hawassa and Lake Ziway was bought
from the informal markets around the shorelines of each lake, between 8.00 a.m. and 9.00 a.m.
local time. They were separately packed in sterile plastic bags with ice blocks to prevent sample
deterioration on transit and transferred to the Addis Ababa University, Institute of Biotechnology,
Health Biotechnology laboratory for bacterial isolation and identification assays.
2.4. Bacterial Isolation and Identification
2.4.1. Sample preparation
All bacteriological experiments were performed following Society of American Bacteriologists
Manual of microbiological methods [11], Bergey’s manual of determinative bacteriology [12]
and the respective manufacturer's instructions under complete aseptic conditions. Using sterile
forceps, a piece of the body, muscles of the fishes were inoculated separately into McCartney
bottles containing selenite-f-broth and Gram-negative broth (HIMEDIA, India) for the isolation
of Salmonella spp. and Shigella spp. respectively, and incubated aerobically at 37ºC for 24 hours.
A loop of the inoculated selenite-f-broth and Gram-negative broth was streaked on a plate of
MacConkey agar (HIMEDIA, India) and incubated aerobically at 37 ºC for 24 hours. Suspected
bacterial colonies were then further picked up with a sterile loop and spread on Desoxycholate
citrate agar medium (HIMEDIA, India) and incubated aerobically at 37ºC for 48 hours. These
colonies were further purified by repeated subculture on nutrient agar. Pure isolates were stored
at -20°C in 50% glycerol (Fine Chemical, Ethiopia) using 1.8ml cryovial (IMEC, China) for
biochemical identification.
2.5. Biochemical characterization
All the media used in the biochemical tests were from HIMEDIA, India. The tests began with
inoculating respective media with 24-hour-old pure culture colonies. All incubations were at
37°C for 24 hours and expected color changes confirmed test positivity. Briefly, indole
production was tested by inoculating 10ml of Dev tryptophan broth, incubating and adding 2-3
drops of indole reagent. Methyl red (MR) test was conducted by inoculating 10ml of MR Voges-
Proskauer (MR-VP) medium, incubating, and adding 2-3 drops of 0.05% MR. Voges-Proskauer
(VP) test was done by inoculating 10ml of MR-VP medium, incubating, and adding 2-3 drops of
5% α-nephtol followed by 40% of KOH and shaking and leaving it open for an hour.
For catalase enzyme test, a small amount of bacterial colony was transferred to a clean glass slide
using a sterile loop and a drop of hydrogen peroxide was added, and the formation of bubbles
was checked for. For citrate utilization test, Simmons citrate agar slant was inoculated and

4
incubated. Hydrogen sulfide or triple sugar iron (TSI) test was done by inoculating TSI by first
stabbing through the center of the medium to the bottom of the tube and then streaking the
surface of the agar slant, and incubating. Similarly, urea production was tested using
Christensen’s Urea Agar slant. Sugar fermentation test was conducted using sugar broth medium
prepared by mixing 1g peptone, 0.3g meat extract, 0.5g table salt, 0.5g sugar and 0.008g phenol
indictor in 100ml distilled water. Three tubes having three different sugars (glucose, sucrose,
lactose) in the broth medium were inoculated, and incubated.
2.6. Molecular Characterization
The bacterial isolates that were biochemically characterized and identified as S. enterica and S.
flexneri were to further identify and confirms using molecular techniques. These involve
extracting their DNA using standards protocols, amplifying the DNA using PCR, and sequencing
the gene of interest using Sanger sequencing at Leiden, The Netherlands [13].
2.6.1. DNA Extraction
DNA was extracted using phenol chloroform standard method as described by Biggin et al. [14].
Bacteria cells each of 200μl was added in a 1.5ml micro centrifuge tube, 400μlof lysis buffer and
10μl of proteinase K was added to the 1.5ml tube. The tube was vortexed and placed on heat
block at 65℃ for 1 hour while vortexing at interval. Then 400μlof phenol was added and
vortexed, briefly after which it was centrifuged for 10 minutes at 13,000rpm to separate the
phases. The upper layer was carefully removed with a micro pipette. Chloroform (400μl) was
again added and vortexed, centrifuged for 5 minutes at 13,000rpm, the upper layer was then
carefully removed. Also 1000μl of 100% ethanol and 40μl of 3M sodium acetate was added and
mixed by inverting the tube several times.
The micro centrifuge tubes containing pure DNA were incubated at 20℃ over night. The tubes
were centrifuged for 5 minutes at 14,000rpm using centrifuge set at 4 o
C; the upper layer was
attentively discarded using micropipette. Then 400μl of 70% ethanol was added and the upper
layer was again carefully removed using micro pipette, the tubes were centrifuged for 5 minutes
at 14,000rpm using centrifuge set at 4o
C, the upper layer was carefully removed using micro
pipette in order to remove all traces of ethanol, the DNA was then allowed to dry by leaving the
tubes open for 20 minutes at room temperature and 50μlof sterile water was added to re-suspend
the DNA.
2.6.2. PCR Amplification
It was performed in a DNA thermal cycler (Eppendorf, Hamburg, Germany). PCR reactions were
performed in a final volume of 25μl containing 3μl of template DNA, 2μl of each primer (rD1
and fD1 ), 14μl of Double distilled water (ddH2o), 2.5μl of Buffer solution, 2μl of MgCl2 and
1.5μl of Hot Star Taq DNA polymerase and deoxynucleotide triphosphate mix (dNTPs). PCR
conditions were as follows: 95 °C for 15 min, 30 cycles at 95 °C for 45 s, 55 °C for 45 s, 72 °C
for 1 min, followed by a final elongation at 72 °C for 7 min. The PCR products (8 μl) were
analyzed by electrophoresis on 1.5% agarose gels stained with ethidium bromide using 1 μM
Tris-Acetate-EDTA buffer at 100v for 1 hour and were visualized by UV transillumination [13].
2.6.3. 16S Rrna Sequencing
Sequencing of the 16S rRNA was performed using the universal bacteria primers rD1 (5'
-
CCCGGGATCCAAGCTTAAGGAGGTGATCCAGCC-3'
) and fD1 (5'
-
CGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG-3'
) by Sanger sequencing at

5
BaseClear DNA research laboratory, Leiden, The Netherlands. For sequencing of amplified 16S
rRNA directly, four identical 100µl amplification reactions were performed on each sample, with
the resultant material being pooled and purified. A 500 ng amount of template (16S rRNA) was
combined with 10 ng of primer, 2µl of Sequence buffer, and 10µl water. This sample was held at
98°C for 7 min and cooled to room temperature for 1 min, and then the labeling reaction was
performed at 37°C for 5 min. Chain elongation was terminated with sample loading buffer, and
sequencing was performed on buffer-gradient gels [14].
2.6.4. Phylogenetic Analysis
The 16SrRNA gene sequences obtained were compared and aligned with sequences deposited in
National Center for Biotechnology Information (NCBI) GenBanks database using BLAST for
identification of bacteria [15]. Related sequences were obtained and aligned by Pairwise
sequence alignments in MEGA6 program using ClustalW algorithm and phylogenetic tree was
constructed by the neighbour-joining method program available online [16]. The 16S rRNA
sequences of isolated bacteria were used for constructing phylogenetic tree. A total of 500
bootstrapped values were sampled to determine a measure of support for each node on the
consensus tree.
2.7. Data Analysis
Bacterial infection status of the fresh fish products sample types of the two lakes was determined
and the proportion of infected samples was compared between various categories using the Chi-
squared test. Bacterial prevalence in the two different sample types was compared using one-way
analysis of variance (ANOVA). Statistical analysis was performed using IBM SPSS software
version 26 (IBM, Chicago, USA) and p<0.05 was considered the level of statistical significance.
3. RESULTS
3.1. Collected Fresh Fish Products
A total of 60 (30 from each) fresh fish products samples were bought and obtained from the
informal market along the two lakes shoreline of which 33(55%) were positive for S. enterica and
S. flexneri pathogens. Among the 55% positive samples, 28.33% (16.67% Nile tilapia fish and
11.67% common carp fish) and 26.67% (13.33% common carp fish and 13.33% Nile tilapia fish)
of the fresh fish products were bought along the shoreline of Lake Hawassa and Lake Ziway
respectively.
3.2. Colony Morphology of the Isolates
Colonies of the isolated isolates were found to be different in their form, elevation, margin,
surface, colour and optical characteristics, and are Gram negative, rod-shaped, usually motile,
non-spore forming bacteria.
3.3. Biochemical Identification of Isolates
To further identify the morphologically identified isolates, a range of biochemical tests (Indole,
methyl red, Voges-Proskauer (VP), Citrate, H2S gas, Urease, sugar fermentation and catalase test)
were carried out. The results showed that thirty three (33) isolates of S. enterica (18) and S.
flexneri (15) were identified.

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3.4. Prevalence of Isolates Among the Examined Fresh Fish Products
Salmonella enterica (30%) was relatively more prevalent than S. flexneri (25%) in the examined
fresh fish products samples. The highest numbers of S. enterica (33.33%) were recovered from
the Lake Hawassa fresh fish products samples followed by Lake Ziway (21.2%).But, the highest
numbers of S. flexneri (27.3%) were recovered from the Lake Ziway fresh fish products samples
followed by Lake Hawassa (18.2%). However, even though highest numbers of both pathogens
were detected from fresh fish products of Nile tilapia than common carp samples (Table 1), their
prevalence showed statistically no significant difference in the samples source of the study lakes
(p<0.005).
Table 1: Frequency of S. enterica and S. flexneri among the examined Nile tilapia and common carp fish
products from Lakes Hawassa and Ziway (N = 33)
Bacteria
isolates
Lake Hawassa Lake Ziway Total P-
value
C. carp Nile tilapia C. carp Nile tilapia
S.
enterica
5(15.15%) 6(18.2%) 3(9.1%) 4(12.12%) 18(54.54%) 0.062
S.
flexneri
2(6.1%) 4(12.12%) 5(15.15%) 4(12.12%) 15(45.45%) 0.096
Sub-total 7(21.2%) 10(30.30%) 8(24.24%) 8(24.24%) 33(100) 0.056
Total 17(51.5%) 16(48.5%) 33(100) 0.192
Note: - C. carp=Common carp
3.5. Analysis of the 16S Rrna Gene
The results of the genomic DNA extraction and PCR amplifications of the samples on agarose gel
electrophoresis shows the amplified 16S rRNA gene with band size of 1490bp obtained from
fishes in two different informal markets location along the two lakes as depicted in Fig. 2.
Fig. 2: Agarose gel electrophoresis pattern showing PCR amplified products of 16s rRNA from S. enterica
and S. flexneri
1490bp

7
3.6. Phylogenetic Analysis
This result represents the blast result of 16S rRNA gene sequences for isolated S. enterica and S.
flexneri. The 16S rRNA ribosomal PCR amplified products sizes were 1490bp. Comparison of
the S. enterica and S. flexneri isolates with known 16S rRNA sequences in the GenBank database
using the BLAST program showed that the isolates had an identity rate of up to 99% to those of
other members of S. enterica and S. flexneri. The phylogenetic relationship of this isolates was
studied using the 16S rRNA gene with the respective S. enterica and S. flexneri strains that were
downloaded from NCBI for phylogenetic analysis (Fig.3).
Fig. 3: The phylogenetic relationships based on 16S rRNA gene sequences of S. enterica and S. flexneri
strains constructed by the neighbor-joining method showing homology with different sources of S. enterica
and S. flexneri. Numerals at nodes indicate bootstrap percentages derived from 500 replications and
original isolates were indicated by bold dot.
4. DISCUSSION
Assessing the occurrence of prevalent etiological agents in fish products is necessary to control
food-borne diseases of fish origins. The result of this study confirms the contamination of fresh
fish products by S. enterica and S. flexneri pathogens. Results obtained revealed the prevalence of
S. enterica and S. flexneri in fresh fish products bought from the informal market along the
shorelines of the two lakes. The results showed that 18 isolates of S. enterica and 15 isolates of S.
flexneri were isolated and identified from the body, muscles of both common carp and Nile
tilapia fish. Similarly, Coulombe and Tamber [17] isolated S. enterica from fish and fish
products. The some authors indicated that, fish and fish products are an important vehicle for
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food borne S. enterica and as this pathogen is the second leading food borne diseases in the
world. Kousar et al. [18] and Onyango et al. [19] on the other hands reported the isolation of S.
flexneri from different organs of apparently healthy and naturally infected fish of different
species including Common carp and Nile tilapia.
Hidayah et al. [20] recorded Salmonella and Shigella from fresh fish product and smoked fish.
The some authors suggested that fish products are among the foodstuffs that are prone to
pathogenic microbial contamination which can cause food poisoning and disease in consumers.
This result also agrees with the results of Ucak and Afreen [21] that showed the contamination of
aquaculture products with salmonella spp., and Shigella spp., and as salmonella and Shigella are
the main causing agents of infection in fish. Nisa et al. [22] also documented the isolation of S.
flexneri from water, milk and fish products, and their association with an increased human risk
to food-borne shigellosis.
Hazarika et al. [23] described as the consumption of food of animal origin including fish products
are considered to be the primary source of human salmonellosis and shigellosis serving as the
principal vehicles of human food borne salmonella and Shigella. The some authors suggested the
prevalence of salmonella spp. and Shigella spp. in different sectors of the global food chain. Non-
typhoidal Salmonella accounted for more than 60% of the food borne disease burden[24]. Dissasa
et al. [25] also indicated the prevalence of salmonella spp., and Shigella spp. in fish, fish products
and the natural source habitat.
Shigella spp. and Salmonella spp. are pathogenic bacteria found in animal, human or
environmental reservoir. Although contamination of fish products with these bacteria is
commonly from the environment, their incidence in ready-to-eat fish products due to unhygienic
handling cannot be ruled out [26]. The presence of Salmonella and Shigella in fish could be due
to microbiological pollution of rivers, including runoff and storm-water that contain deposits
from wildlife, agriculture, urban, forestry and rural settlements which is a potential health hazard
to human; another possibility includes contamination during a multistep handling process from
harvest areas to the fish market resulting in deterioration of the quality of fish available for
human consumption in the local fish markets [27].
Sources of S. enterica and S. flexneri infection can vary by environment and include animal and
plant-based foods, as well as environmental reservoirs. Several studies have recognized the
importance of increased ambient temperature and precipitation in the spread and persistence of
Salmonella in soil and food [28]. Primary production of food animals remains the most important
reservoir of salmonellae and Shigella entering the human food chain, since salmonellae and
Shigella-free production system cannot be achieved in all animal species. Controls at slaughter
and dressing are often not sufficient to prevent salmonella and Shigella entering the food chain.
The prevalence of Salmonella in fish captured from aquaculture is relatively lower than in
freshwater captured fish, due to possible exposure to untreated sewage, but overall, and in
comparison with other food commodities, both remain low risk [29].
5. CONCLUSION
Fish are considered as a nutritious food that is widely produced, marketed, and consumed around
the world. Nile tilapia and common carp are among the fish with the highest production, destined
for human consumption. Fish are highly susceptible to contamination and deterioration along the
food chain by different microorganisms, such as Salmonella enterica and Shigella flexneri, which
is a causal agent of disease in fish, such as Nile tilapia and common carp, and humans negatively
affecting primary production and food safety. The issue of Salmonella and Shigella in fish and
fish products is one of the global concerns. The difficulty in eliminating S. enterica and S.

9
flexneri to safe levels in fish products underscores the importance of preventing contamination.
For S. enterica and S. flexneri, which are common in natural water systems and is considered as
part of the microbiota of freshwater fish such as Nile tilapia and common carp, the
implementation of good hygiene practices in production, processing and preservation, and
sanitary surveillance of regulatory authorities and the food industry, as well as the promotion of
hygiene education in the handling and final preparation of these foods for consumption, can
reduce the risk of contamination and related outbreaks of food borne diseases.
ACKNOWLEDGEMENT
The authors are grateful to the fishermen selling fresh fish products along the shoreline of the two
study Lakes for their technical assistance during sample collection.
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