1. The Intensities of Escherichia coli and Pseudomonas Bacteria in Oreochromis niloticus from Kesses Dam and
University of Eldoret Fish Farm
Lusega D. Manguya*
Department of Fisheries and Aquatic Sciences
University of Eldoret, Kenya
dmanguya@yahoo.com
Nabwire Electine
Department of Fisheries and Aquatic Sciences
University of Eldoret
tynamwaniga@gmail.com
*Corresponding author
ABSTRACT
Human infectionscaused by pathogenstransmitted from fish are currently common. This study focuses on the level of
bacterial counts(Escherichia coliand Pseudomonas spp) in fresh Nile Tilapia(Oreochromis niloticus) harvestedfrom
University of Eldoret and Kesses dam in Uasin Gishu county. It also gives a comparison of prevalence of the two
bacterial strains in fish from the two sources. Thirty (30) fresh samples were collected from each source and
transported to the laboratory in a clean disinfected cool box for analysis. Equipment used were first sterilized in an
autoclave at 120°C for thirty minutes before use. A representative and homogenous sample was taken from the skin,
gillsand gut ofthe fish for analysisusing the swabbing method.After culture of the two bacteria colonies were counted
using a colony counterand theirnumbers recorded. The results obtained were analyzed using Chi-square.There was
significant difference in the bacterial prevalence in the two sources where Kesses dam had high percentages of the
two bacteria. The fish were also found to have the two bacteria above the recommended microbiological standards
for food fish hence posing a health risk to the consumers. The presence of these bacteria is attributed to the
contamination of the dam and the farm with faecal matter of human and animal origin. Surface runoff during rainy
seasons, source of water used in UoE farm and the rivers flowing into Kesses dam may be responsible for the
environmental pollution since they carry along sediments containing the bacteria and both organic and inorganic
matter. The study recommends that the water used in UOE farm be treated before use, drainage systems constructed
to contain surface runoff and animals e.g. sheep, goats among others kept off the farm. Equipment used in the farm
should be disinfected regularly and feeds kept under proper hygienic condition to avoid contamination. Consumers
who obtain fish from Kesses dam are advised to cook the fish properly to ensure the bacteria are destroyed before
consumption since it’s not possible to prevent pollution of the dam. Further research on the same is recommended
too.
Key Words: Escherichia coli, Pseudomonas Bacteria, Oreochromis niloticus
INTRODUCTION
Nile tilapia (Oreochromis niloticus) is the most widely bred fish species in the world. This can be explained by some
relevant features of this species, such as fast growth rate, high-quality meat and good flavor, which lead to its good
acceptance by consumers. FAO asserted that fish contributes about 60% of the world supply of protein and that 60%
of the developing world derives more than 30% of their animal protein from fish. Fish allows for protein improved
nutrition in that it has a high biological value in terms of high protein retention in the body, low cholesterol level and
presence of essential amino acids. In recent times there has been renewed effort in fish production especially in
artificial (concrete and plastic) ponds as a way of augmenting the protein requirement of the increasing human
population in urban areas with the prohibitive price of beef and other animal protein sources (Emikpe et al., 2011).
Tilapia offers the possibility of commercial and home-grown protein sources where wild capture fisheries are being
depleted.
Currently there is scarcity of information on microbial spoilage of fresh fish. The last two decades have seen
appreciable increase in global fish trade and the need to enforce safety standards and regulations on imported
consignment especially from developing nations fraught with unacceptable levels of microbiological contamination
concern is on high loads of unsuspected spoilage by microorganisms like Salmonella sp., Staphylococcus aureus,
Pseudomonas aeruginosa, Escherichia coli among others.

2. In every country where fish inspection programme exists, the load of faecal coliforms in farmed, feral or processed
fish is evaluated to verify whether the harvest orproduct presents a health hazard ornot.Their presence in fish intended
for human consumption may constitute a potentialdanger not only in causing disease,but also because ofthe possible
transfer of antibiotic resistance from aquatic bacteria to human infecting bacteria from non-aquatic sources. Some
bacterial species used as indicators in monitoring environmental quality include; Coliforms, E.coli, Streptococcusspp,
Pseudomonas spp, Vibrio spp, Clostridium spp, Bifidobacterium pseudolongum, Arcobacter spp, Thiobacillus spp
among others.
If these indicator bacteria are present, there’s a probability that pathogenic micro-organisms (Bacteria, Viruses and
Protozoa) excreted in faeces are present and that water can transmit waterborne infectious diseases, Shawky, 2005.
According to Emikpe et al 2011, Fish and Shellfish not only transmit disease to man but are themselves subject to
many diseases and capable of transmitting many of the established food borne microbial infections and intoxications.
The microbiology of fish skin and gastro intestinal tract has been subjected to many researches. Fish can spoil from
both outer surface and inner surfaces as fish stomach contains undigested and partially digested food which can pass
into the intestine. After fish has been caught and dying the immune system collapses and bacteria are allowed to
proliferate freely on the skin surface and the stomach. The walls of intestines do break down sufficiently for bacteria
to move into the flesh through the muscle fibre. It has been suggested that intestinalmicro flora is the causative agent
for food spoilage. Contamination of fish from enteric bacteria of human and animal origin may also be responsible
for various food spoilages.
Invasion of fish muscle due to the breakage of immunological barrier of fish by pathogens is likely to occur, when the
fish are raised in pond with faecal coliforms, E. coli and of greater than 103 per ml (Guzman et al., 2004). Fish take a
large number of bacteria into their gut from water sediment and food. It has been well known that both fresh and
brackish water fishes can harbor human pathogenic bacteria particularly the coliform group. Faecal coliform in fish
demonstrates the level of pollution in their environment because coliform are not named flora of bacteria in fish. The
ponds and rivers that harbour the fish may be the source of contaminates due to indiscriminate deposition of human,
animal excreta and other environmental wastes into natural water, land and during the rainy season especially, as the
faecal matter from various sources are washed fromcontaminated land into different water bodies.
Free roaming animals and pets especially dogs also contribute to faecal contamination of surface water. Run -off from
roads, parking lots and yards can carry animal wastes into natural water course and ponds. Birds can also be a
significant source of bacteria. Swans; Geese and other water fowl can all elevate bacteria counts in water bodies and
ponds.
Microbiological control in food production is an important aspect in the supply of safe, nutritious and palatable food
with adequate shelf life. This refers to the absence, exclusion or elimination of pathogenic micro-organisms.
Pseudomonas, Shigella, Salmonella, Klebsiella and E.coli are among several bacterial strains responsible for more
than 40,000 cases of food-borne illness every year (FAO 2010). Incidences of food/waterborne infections has risen
since the 1990s, leading to high medical costs,loss of wages for workers who become ill, and a loss of productivity
for the companies whose workers do become ill . In all, these financial losses can cost more than $3.6 billion each
year which could be invested elsewhere; the bacteria in large numbers may cause spoilage of fish hence economic
losses too. Most strains of E.coli are resistant to antibiotics and the resistance may be transferred to humans, some
cause dysentery too. Pseudomonas on the other hand is known to cause nosocomial infections and ability to destroy
tissues too. Fish is known to harbour majority of bacteria due to the nature of their environment. Nile Tilapia has
recently been on high demand hence there is a risk of transmission of these bacteria to the consumers in case they
exceed the recommended standards (Sichewo et al., 2013). Adebisiet al (2011) carried out a similar research in South
West Nigeria where Clarias gariepinus from two different sources were used. They termed the current conditions
used for culture of fish as a major contributorto the increase of bacterial prevalence in fish. A similar study conducted
by Onyango et al, 2009 in Nile Tilapia from several landing sites around Winam Gulf in Kisumu, Kenya indicated

3. that E.coli was the most prevalent bacteria in fish and above the recommended standards. In the study the cases of
infections due to this bacteria was 32% hence alarming. This study therefore is meant to investigate the presence of
Escherichia coli and Pseudomonas Bacteria in Oreochromis niloticus from Kesses Dam and University of Eldoret
Fish Farm, whether they comply with the set standards and if they have an impact on the health of the consumers
around Eldoret.
METHODS USED
The samples were caught from University of Eldoret (UOE) fish farm using seining method where monofilament nets
were used and in Kesses Dam fish were caught by the fishermen using monofilament nets too. 30 fresh Nile Tilapia
samples were obtained from each source. Samples were washed with clean distilled water and transported to the
laboratory in a clean sterilized cool box for analysis. Before the analysis all glassware and the media (Mac-Conkey
and Eosin Methylene Blue agar) were sterilized in an autoclave for thirty (30) minutes at a temperature of 120°C while
other equipment of metallic nature were sterilized in an oven for ten (10) minutes at a temperature of 750°C. A
representative and homogenous sample was taken from the gut, skin and gills of the fish for analysis. The analysis
was carried out under an air filter to avoid further contamination of the samples. Spread method was used to cu lture
the bacteria in their specific media, all the petri-dishes containing the samples and the media were covered using a
parafilm before being transferred to the incubator set at 37°C for 24 hours for E.coli and 48 hours for Pseudomonas
spp. Bacterial colonies were counted in the plates using a colony counter and recorded.
Microbial Analysis
Skin surface and gut: A sterilized wire swab guide measuring was placed on the lateral surface of the fish sample.
The cotton wool swab was dipped in 0.10% sterile peptone water and rubbed over the surface of the fish on the area
covered by the wire swab guide. The swab was then placed in a sterile sample bottle containing 10mls of 0.10% (w/v)
peptone water.The bottle vigorously shaken for10 mins and allowed to stand for20 mins. Three (3) fold serial dilution
of the bacterial suspension in peptone water was prepared in duplicate and viable bacterial counts enumerated in
standard plate count agar after incubation at 370C for 48 hrs.
Gills: Ten grams of the fish gills sample was dissected out blended and mixed properly. It was ascetically transferred
to a sample bottle containing 9 mls of 0.1% sterile peptone water.The bottle was closed and shaken thoroughly for 10
mins and allowed to stand for 20 mins.
A three (3) fold serial dilution was carried out in duplicates and viable aerobic bacterial counts were enumerated in
standard plate count agar after incubation at 37OC for 48hrs as described by Slabyet al. (1981).
Escherichia coli count: Volumes of 1 ml from undiluted,1/10 and 1/1000 dilutions were spread on Eosine Methylene
Blue Agar in duplicates and incubated at 37oC for 24h to enable growth of bacterial colonies. Colonies with green
metallic sheen were counted as E. coli after the culture period.
Pseudomonas sp: Volumes of 1ml from undiluted, 1/10 and 1/1000 dilutions were spread on Mac-Conkey agar in
duplicates and incubated at 37°C for 48hours where circular mucoid, smooth colonies were counted as Pseudomonas
sp.Fewer than 30 colonies are not acceptable for statisticalreasons (too few may not be representative of the sample),
and more than 300 colonies on a plate are likely to produce colonies too close to each other to be distinguished as
distinct colony-forming units (CFUs). The assumption is that each viable bacterial cell is separate from all others
and will develop into a single discrete colony (CFU). Thus,the number of colonies should give the number of bacteria
that can grow under the incubation conditions.
Data Analysis: Data and results were statistically computed using Minitab 14. The colony forming units (cfu) per
10ml/1gm were being converted to log10 colony forming units (logcfu)/gm of sample for subsequent data analysis.
The comparison of bacterial load from the two sources (Kesses and UOE farm) was determined using Chi-square
analysis.

4. RESULTS
The following results were obtained from the Chi-square test which was used for analysis; the results give a
comparison of the bacterial abundance in various parts of the fish sampled from the two sources (Kesses dam and
UOE farm).
Gills
Kesses -70.60%
UoE farm-29.40%
Pearson Chi-Square = 15.847, DF = 2, P-Value < 0.0001
Likelihood Ratio Chi-Square = 15.787, DF = 2, P-Value < 0.0001
Cramer's V-square 0.0004936
Gut
Kesses -71.39%
UoE farm-28.61%
Pearson Chi-Square = 13.816, DF = 2, P-Value < 0.001
Likelihood Ratio Chi-Square = 13.678, DF = 2, P-Value < 0.001
Cramer's V-square 0.00089
Skin
Kesses -68.92%
UoE farm-31.08%
Pearson Chi-Square = 15.847, DF = 2, P-Value < 0.0001
Likelihood Ratio Chi-Square = 15.787, DF = 2, P-Value < 0.0001
Cramer's V-square 0.0004936
Measures of Association
From the measures of association tests,the pairs were 100% concordant indicating that there is an association between
the source of the fish and the amount of bacterial colonies isolated. Measures of Association usually have values
ranging from 0-1 where values close or equal to one indicate that the model has a better predictive ability.
In this case the Sommers’ D test and the Goodman kruskal Gamma tests both have values of 1 indicating that the
model has a better predictive ability hence suitable in this case.
Compliance with the set microbiological standards of food fish
According to the EU the microbiological standards of fresh fin fish should comply with the following;
E.coli; 0
Pseudomonas sp; 0
Pseudomonas sp
UOE farm; 100% above the standards.
Kesses dam; 100% above the standards.
E.coli
UOE farm; 100% above the standards.
Kesses dam; 100% above the standards
Fish from the two sources had bacterial counts above the set microbiological standards by the European Union of the
two strains. The local standards have been meet by some of this fish where Pseudomonas sp<5×101 are acceptable
hence 90% of fish from UOE farm are below these standards while only 10% of fish from Kesses dam meet the
standards.
In compliance with ourlocal E.coli standards (<100/g of fish), 70% of fish from UOE farm comply with this standards
while 25% of fish from Kesses dam comply. From these results, a large percentage of fish from Kesses dam have
bacteria above the local standards as compared to those from UOE farm where a large percentage are below the
standards.

5. DISCUSSION
Gills: The Pearson chi-square p-value of 0.0001, the Likelihood Ratio Chi-square p-value of 0.0001 and Cramer’s test
with 0.0004936 indicate that there is a significant difference in the abundance of bacteria in the gills of fish from the
two sampled areas of study.Kesses damhad the highest percentage ofbacteria in gills (70.60%) while UOE farm had
29.40%.Presence of bacteria in the gills is attributed to their function where water carrying these bacteria pass through
the gills for gaseous exchange hence the bacteria get attached to the gills easily.
Gut: From the Chi-square and associate tests carried out, the p-values (0.001, 0.001 and 0.00089) indicate that there
is a significant difference in the abundance of bacteria in the gut of fish from the two sources.
Kesses dam however indicates a high percentage of bacteria (71.39%) as compared to UOE farm (28.61%).The
presence of bacteria in the gut of fish can be attributed to the fact that food consumed by the fish passes through the
gut for digestion.
The food may acquire the bacteria from the water column, poor preparation of the food under unhygienic conditions
and unhygienic storage of the feeds may lead to their contamination by the bacteria.
Skin: According to the p-values obtained from the test,(Pearson test; 0.0001, Likelihood Ratio; 0.0001 and Cramer’s
V-square test; 0.0004936) there is significant difference in the abundance of bacteria isolated from the skin of fish
from the two sources.Kesses damgave the highest percentage of bacteria isolated from the skin (68.92%) compared
to UOE farm(31.08%)The skin of fish is highly exposed to bacteria in their environment hence their presence indicates
the level of pollution in the system. Bacteria in the dam is as a result of surface runoff draining into the dam and the
rivers flowing into the dam too. They both carry along both organic and inorganic pollutants, faecal matter of both
human and animal origin and sediments that could harbour these bacteria into the dam. The UOE’s source of water
could be the source of bacteria since water in not disinfected before being used.Surface runoff too could carry along
the bacteria into the culture systems.
The farm equipment contributes to the presence of bacteria when they are poorly stored or handled, being used in
several ponds without disinfecting could transfer bacteria from one pond to the other too
The presence ofbacteria in the two sources is attributed to various factors surrounding the environments. In UoE farm
the bacteria may be carried into the ponds through surface runoff during rainy seasons,the source of water may also
be contaminated with the bacteria of faecal nature since the water in not treated or disinfected before being let into the
ponds.There is also an evidence of some animals roaming around the farm these include goats and sheep which could
be a possible source of bacteria through their wastes. Poor handling of farm equipment where one net is used in
different ponds without prior disinfection may be responsible for transfer of bacteria among the ponds, unhygienic
preparation and storage of feeds which may lead to their contamination with bacteria of faecal nature which are
consumed along with the feeds by the cultured fish hence their multiplication within the gut of the fish.
In Kesses dam, monitoring of the inlets is impossible hence the two rivers (Tarakwa and Nderugut) which feed the
dam highly contribute to the high invasion of bacteria in the dam in addition to the surface runoff during rainy seasons.
Both organic and inorganic wastes are washed into the streams flowing to the dam with human and animal wastes
being the major contributors of bacteria into the water. The fact that the volume of water is high, the impossibility of
treating the water is enough forfurther multiplication of the bacteria at a fasterrate due to the condusive environment.
There was evidence of further pollution by the residents of the area washing clothes and utensils around the dam and
some animals roaming around too. The presence of bacteria in the two sources as seen in the results therefore is an
indication of pollution in the two places with faecal matter.
CONCLUSION
Fish from Kesses damharbour more bacteria than those from University of Eldoret Fish farm, this is attributed to the
nature of the two environments where Kesses damreceives large amount of water from surface runoff due to the large
area it covers hence more sediment and bacteria carrying organic and inorganic material are washed away into .the
dam. Kesses damis also fed by two different streams which carry along a lot of sediments,animal and human wastes
among other wastes hence the pollution level is quite high as compared to UOE farm.

6. From the study the bacterial load exceed the recommended standards of bacteria in fresh fish hence the presence of
these bacteria isolates is indicative of public health risk in contracting diseases associated with these organisms. The
results of this study are in line with results of a similar study conducted in West Nigeria by Adebisi et al. (2011), in
Bangladesh by Shankar et al, 2009 where both studies compared bacterial loads of faecal coliforms, spoilage bacteria
and other pathogenic bacteria in cultured fish and fish from the wild. In both studies, fish from the wild had had a
higher prevalence of all the bacteria isolated as compared to cultured fish. In another study conducted in Winam Gulf
of Lake Victoria, Kenya by Onyango et al,2009, who isolated Salmonella sp from fish obtained from the landing sites
of the study area. These bacteria were found to be above the recommended standards and they concluded that this
isolation was as a result ofcontamination of the waters by the pathogen posing a health riskto the consumers.Onyango
et al. (2009) took a survey study too on the numbers of patients treated against foodborne illnesses that maybe
transmitted through consumption of affected or infected fish. The survey results indicated that 5.2% of residents were
treated in 2009 following diarrheal infections caused by Enterobacteriaceae.37.5% were diagnosed with E.coli related
infections around Kisumu town and 6.8% around Uhanya in Bondo district. This therefore indicates that these bacteria
may pose a health risk to consumers if not well managed.
RECOMMENDATION
The presence ofdiverse enteric bacteria in the aquaculture environments suggests that strict hygiene procedures should
be followed during the handling and processing of fish from the culture systems to prevent the transfer of potentially
pathogenic bacteria to humans. Thus there is need for a code of practice for fish growers in tropical aquaculture
systems to ensure safe food sources.
For fish from the wild where it’s difficult to prevent invasion of bacteria it’s important for the consumers to cook
properly before consumption to ensure the bacteria are killed since at high temperatures for long periods of time the
pathogen will not survive.
Further research should be conducted to find possible ways of containing these bacteria in natural waters to prevent
invasion and on the microbiological standards since ourlocal standards highly differ from the globally used standards
of the EU.
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