THE BACTERIAL FLORA OF TILAPIA (OLEOCHROMIS NILOTICUS) AND CATFISH

(CLARIAS GARIEPINUS) FROM EARTHEN PONDS IN SAGANA FISH FARM AND

MASINGA DAM

ROSALINE DAISY KARIMI

REG. NO. 156/7076/2001

A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR

THE AWARD OF THE DEGREE OF MASTERS OF SCIENCE (INFECTIOUS

DISEASE DIAGNOSIS) IN THE SCHOOL OF PURE AND APPLIED SCIENCES,

KENYATTA UNIVERSITY

MAY 2015

i
 DECLARATION

This thesis is my original work and has not been presented for a degree in any other University or any

other award.

Rosaline Daisy Karimi

Signature………………… Date…………………………

This thesis is submitted with our permission as University Supervisors

Prof. Joseph J.N. Ngeranwa

Department of Biochemistry and Biotechnology

Kenyatta University

Signature……………………… Date………………………….

Prof. Eliud N.M Njagi

Department of Biochemistry and Biotechnology

Kenyatta University

Signature……………………… Date………………………….

Dr. Sam Kariuki

Centre for Microbiology,

Kenya Medical Research Institute

Nairobi Kenya

Signature……………………… Date………………………….

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 DEDICATION

I dedicate this thesis to God the almighty for his mercies and strength to persevere to

the end of the work. To my loving husband Charles, my children Cynthia, Fiona and

Allan for their love, endurance and support.

iii
 ACKNOWLEDGEMENTS

This work would not have been achieved without the tireless effort of my supervisor,

Prof. Joseph J.N. Ngeranwa and Prof. Eliud N.M. Njagi from Kenyatta University

and Dr. Sam Kariuki from KEMRI for their encouragement, advice, support and

professional guidance offered in supervising the work from its inception to its

completion. I wish to express my gratitude to the Director KEMRI for allowing me

to carry out my research at their laboratory. My deep appreciation goes to the

laboratory technicians at KEMRI in particular Ms Agnes Kanini, who worked

closely with me.

My heartfelt gratitude to my family for the patience and perseverance endured during

the preparation of this thesis. I am indebted to my loving husband and friend Muriuki

Njagagua for the financial support throughout the entire period. To my children

Cynthia, Fiona and Allan, thank you and may God bless you abundantly.

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 TABLE OF CONTENTS

DECLARATION ......................................................................................................... ii

DEDICATION ............................................................................................................ iii

ACKNOWLEDGEMENTS ........................................................................................ iv

TABLE OF CONTENTS ............................................................................................. v

LIST OF TABLES ..................................................................................................... vii

LIST OF FIGURES .................................................................................................. viii

LIST OF ABBREVIATIONS AND ACRONYMS.................................................... ix

DEFINATION OF TERMS ......................................................................................... x

ABSTRACT ................................................................................................................ xi

CHAPTER ONE .......................................................................................................... 1

INTRODUCTION ....................................................................................................... 1
1.1 Background Information ........................................................................................ 1
1.2 Statement of the Problem ....................................................................................... 3
1.3 Justification of the study ........................................................................................ 4
1.4 Research Questions ................................................................................................ 5
1.5 Objectives of the Study .......................................................................................... 5
1.5.1 Main Objective .................................................................................................... 5
1.5.2 Specific Objectives.............................................................................................. 5

CHAPTER TWO ......................................................................................................... 6

LITERATURE REVIEW............................................................................................. 6
2.1 Nutritional and economic value of fish .................................................................. 6
2.2 Bacteria associated with farmed fish...................................................................... 6
2.2.1 Vibrio paraheamotyticus and other vibrios.................................................... 8
2.2.2 Escherichia coli ............................................................................................. 9
2.2.3 Salmonella spp ............................................................................................... 9
2.2.4 Staphylococcus aureus ................................................................................. 10
2.2.5 Listeria monocytogenes ............................................................................... 10
2.2.6 Clostridium botulinum ................................................................................. 11
2.2.7 Pseudomomas aeruginosa ........................................................................... 11
2.2.8 Aeromonas species ....................................................................................... 11
2.2.9 Citrobacter freundii ..................................................................................... 12
2.2.10 Edwardsiella tarda....................................................................................... 13
2.3 The Analytical Profile Index (API 20E) .............................................................. 13
2.4 Antimicrobial Resistance ..................................................................................... 14

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2.5 Histamine fish poisoning ..................................................................................... 15
2.6 Factors contributing to fish contamination .......................................................... 16

CHAPTER THREE.................................................................................................... 18
MATERIALS AND METHODS ............................................................................... 18
3.1 Study Site ............................................................................................................. 18
3.2 Sample size........................................................................................................... 19
3.3 Study design ......................................................................................................... 20
3.4 Sampling procedure ............................................................................................. 20
3.4.1 Collection of fish samples ................................................................................. 20
3.4.2 Collection of Water samples ............................................................................. 21
3.4.3 Collection of water sediments ........................................................................... 21
3.5 The Analytical Profile Index (API 20E) .............................................................. 22
3.6 Antibiotic susceptibility testing............................................................................ 22
3.6.1 Antibiotic sensitivity profile ............................................................................. 22

CHAPTER FOUR ...................................................................................................... 24

RESULTS .................................................................................................................. 24
4.1 Bacteria isolates per specimen type ..................................................................... 24
4.1.2 Types of bacteria isolates from Tilapia, Catfish, Water, Water sediments. ...... 24
4.2 Types of bacteria isolates from Sagana pond and Masinga dam ......................... 27
4.2.1 Bacteria isolates from Sagana pond and Masinga dam by specimen type ........ 28
4.3 Bacteria isolated from Tilapia, Catfish, Water and Water sediments .................. 28
in dry and wet seasons................................................................................................ 28
4.3.1 Types of bacteria isolated from the dry and wet season ................................... 29
4.3.2 Bacteria isolates during wet and dry season by specimen type ........................ 30
4.4 Correlation between Bacteria isolates from Specimen types, Site and Season.... 32
4.5 Overall antibacterial response of isolates ........................................................... 33
4.6 Antibacterial response of isolates from Sagana Ponds ........................................ 34
4.7 Antibacterial Response of Isolates from Masinga ............................................... 37

CHAPTER FIVE ........................................................................................................ 39

DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS ........................... 39
5.1 Discussion ............................................................................................................ 39
5.2 Conclusions .......................................................................................................... 44
5.3 Recommendations ................................................................................................ 45

REFERENCES........................................................................................................... 47

APPENDICES ........................................................................................................... 59
Appendix 1: Overall data on specimen, isolates and the response to antibiotics ....... 59

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 LIST OF TABLES

Table 4.1: Bacteria isolates in Tilapia, Catfish, Water and Water sediments ............ 26

Table 4.2: Types of bacteria isolated from Sagana pond and Masinga dam.............. 27

Table 4.3: Number of bacteria isolated from Sagana farm and Masinga dam ........... 28

Table 4.4: Bacteria isolates from dry and wet season ................................................ 30

Table 4.5: Bacteria isolates in specimen type by season ........................................... 31

Table 4.6: correlation between specimen, site and season ......................................... 32

Table 4.7: Antimicrobial Response of Isolates to various Antibiotics....................... 33

Table 4.6 Antimicrobial Response of Isolates from Sagana ...................................... 36

Table 4.7 Antibacterial response of isolates from Masinga dam ............................... 38

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 LIST OF FIGURES

Figure 3.1: Map of Kenya showing Sagana fish farm and Masinga dam .................. 19

Figure 4.1: Bacteria isolates from various collected samples .................................... 24

Figure 4.2: Bacteria isolates in dry and wet season by specimen type ...................... 29

Figure 4.3: Percentage distribution of antimicrobial response of isolates to various 34

antibiotics ................................................................................................................... 34

Figure 4.4: Number of Isolates resistant to antibiotics from Sagana ......................... 35

Figure 4.5: Number of bacterial isolates from Masinga dam resistant to antibiotics. 37

viii
 LIST OF ABBREVIATIONS AND ACRONYMS

API Analytical profile index

DAO Diamine oxidase

DHA Docosahexaenoic acid

EHEC Entrohaemorhagic Escherichia

EPA Eicosapentanoic acid

FAO Food and Agricultural Organisation

FD Fisheries Department – Kenya

HMT Histamine N- methyltransferase

ICMSF International commission on microbiological specifications for foods

NACA Network of Aquaculture Centres in Asia-Pacific

NCCLS National committee for clinical laboratory standards

WHO World Health organization

ix
 DEFINATION OF TERMS

Aquaculture Aquaculture is the farming of aquatic organisms such as fish,

shellfish and even plants. The term aquaculture refers to the

cultivation of both marine and freshwater species and can

range from land-based to open-ocean production.

Capture Fisheries Capture fishery refers to all kinds of harvesting of naturally

occurring living

Pathogenic Bacteria These are bacteria that cause bacterial infection such as

foodborne illnesses.

Finfish A fin fish is another name for true fish. These are fish that

have all the characteristics of fish such as breathing via gills

and their body being covered in scales. The other types of fish

that are not fin fish are called flat fish.

Shellfish This a culinary and fisheries term for exoskeleton-bearing

aquatic invertebrates used as food, including various species of

molluscs, crustaceans, and echinoderms. Although most kinds

of shellfish are harvested from saltwater environments, some

kinds are found only in freshwater

Salmonella roe These are the eggs of the salmon fish cured and used like other

roe products. Like other roes, salmon eggs are collected by

harvesting female fish shortly before spawning, when they

have large and very well developed egg mass.

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 ABSTRACT

Fish is a worldwide distributed food commodity regarded a cheap source of protein

especially in the developing countries like Kenya. It provides a good balance of
protein, vitamins and minerals. However, bacteria occur naturally on the skin, in the
gut and in the slime of living fish, even though they do no harm to the, the
microorganisms may cause harm to consumers. Also, some of the microorganisms
associated with fish may carry genes of antibiotic resistance that can be passed to
pathogens of clinical importance. Food borne diseases traced to fish consumption
have been reported world over including Kenya. In Kenya though aquaculture has
been promoted, the aspect of food quality as far as consumption of fish is concerned
is underestimated. Sagana fish farm and Masinga dam provide fish for Kenyan
markets including Nairobi. No information available when this study was conducted
on quality of fish from Sagana fish farm and Masinga dam. The study was designed
to determine the bacterial flora of Tilapia and Catfish from earthen ponds at Sagana fish
farm and Masinga dam and to determine the anti- microbial response of the pathogenic
bacteria. Samples of Tilapia fish and Catfish were collected from Sagana farm and Masinga
dam in dry and rainy season. The fish were skinned and gut content taken for laboratory test.
Samples of water and water sediments from the two study sites were also collected. The
samples were processed and cultured in MacConkey agar and the colonies sub cultured
in selective media. The colonies were subjected to morphological examination from
cultures and biochemical test carried out using commercially available API kits. The
results obtained from this study showed the presence of bacterial species belonging
to Enterobacter spp. (n=34), Aeromonas spp. (n=5), Vibrio spp. (n=3), Pseudomonas
spp. (n=6) and Acinetobacter spp. (n=2) were isolated during the dry season while
bacterial species belonging to Enterobacter spp. (n=31), Aeromonas spp. (n=4),
Pseudomonas spp. (n=6) were isolated during the dry season. Antimicrobial
susceptibility showed that the highest rates of resistance was found against
Amoxicillin (Aml) (65.9% of isolates), Ampicillin (Amp) (61.5% of isolates),
Tetracycline (Te) (31.8% of isolates), and Chlorampenicol (C) (27.5 % of isolates)
while the lowest was Nalidixic acid (Na), Streptomycin (S) and Cefuroxine (Cxm) at
(4.4% of isolates) each. All isolates were sensitive to Ciprofloxacin (Cip),
Gentamycin (Gen) and Cefotaxine (CTX). The presence of the above organisms
some of which are potentially pathogenic to humans is an indication that fish
improperly handled, undercooked or consumed raw may cause disease to susceptible
individuals while the antimicrobial resistance by some of the isolates is an indication
that the use of antibiotics in aquaculture for promotion of growth should be studied
further with view to policy formulation.

xi
 CHAPTER ONE

INTRODUCTION

1.1 Background Information

Fish is an important component of diets around the world. An estimated 1 billion

people rely on fish as their main source of animal protein (FAO, 2007). The global

fish production is estimated to be 148.5 million tonnes per year of which capture

fisheries accounts for 88.6 million tonnes and aquaculture 59.9 million tonnes per

year (FAO, 2012). Fish provides the much needed protein to people living in

developing countries at affordable prices. It feeds millions of people daily and

sustains many through employment in services related to fish and fish products. The

nutritional attributes of fish are highly praised as it is rich in the essential amino acids, has high

quality vitamins and its fatty acids fraction have well established health benefits (anti-thrombotic

activity). Therefore, its availability in many developing countries should enable fish to

contribute significantly to a healthy and balanced diet in these countries. It is estimated that

around 60 percent of the population in many developing countries derive over 30 percent

of their animal protein supplies from fish, while almost 80 percent of the population in

most developed countries obtain less than 20 percent of their animal protein supplies from fish

(FAO, 2000).

The risks of food borne disease associated with products from aquaculture are related to

inland or coastal ecosystems, where the potential of environmental contamination is greater

when compared with capture fisheries. Most of the food safety hazards associated with

products from aquaculture can be controlled by good fish farm management practices and

appropriate consumer education regarding such risks as eating raw or partially cooked

1
products that may contain pathogenic bacteria (Reilly, 1998). The estimated annual

mortality of food and water-borne infectious diseases in developing countries amounts to high

death rates, mainly of infants and children. In industrialized countries microbiological food

borne illnesses affect up to 30 percent of the population. Every year 20 out of each

million inhabitants die from food borne disease. Unwholesome fish and fishery products

cause up to 30 percent of the food-borne illnesses (WHO, 1999).

Around 40 million people in Asia are affected by fish and water borne parasitic

diseases, especially trematodes. These parasitic diseases are widespread mainly in China,

Viet Nam, Thailand and Laos, where food habits encourage the consumption of raw fish. In

addition to the economic losses incurred because of fish spoilage, fish-borne illnesses can

have costly health adverse effects, the loss of productivity, medical expenses and the adverse

publicity to the companies. Additional costs in international trade include the cost of rejections,

detection of products, recalls and the resulting adverse publicity to the industry and even to the

country of origin of the fish (Lahsen, 2003). Financial implications of food borne disease

outbreaks shows that the consequences can be grave. An outbreak of cholera in Peru in

1991 cost 770 million dollars, a similar outbreak in Tanzania in 1998, cost 36 million dollars.

Simple preventive measures and effective surveillance systems which would cost less might

have prevented these outbreaks, or would have definitely reduced the impact of the (Lahsen,

2003). In Kenya there was ban on exports of fish and fishery to the EU due to an outbreak of

salmonella in 1996 and outbreak of cholera in 1997 (FD, 1999).

In Kenya the future of aquaculture is bright considering that many people are

increasingly turning to fish as a source of their animal protein. With this increase in

demand for fish and the decreasing catches from the natural sources aquaculture is

2
destined to become an important alternative to traditional agricultural practices. The

aquaculture production in Kenya in the year 2008 was 1012 metric tonnes (FD,

2009). In 2011 the figures went high to 24,000 metric tonnes due to the

government’s intervention (FD, 2012). Despite the environmental concerns,

aquaculture profitability is so high that money can and should go back into

promoting sustainable practices. Furthermore, new methods minimize the risk of

biological and chemical pollution through minimizing stress to fish, vaccinating fish,

fallowing netpens, and applying Integrated Pest Management (Paul, 2006).

Current knowledge of the health and environmental impact of antibiotics used in

aquaculture is poor particularly in developing countries. Drug residues may remain

in fish used for human consumption and consequently the antibiotics released into

the environment can lead to the development of antibiotic resistant bacteria in the

food chain (Cabello, 2006). Due to increase in semi-intensive aquaculture systems

there is a potential hazard related to the development of antibiotic resistance in the

pond micro flora. Resistance to antibacterial agents is a major global public health

problem and one that is increasing as these agents continue to lose their effectiveness

(Akinbowale et al., 2006).

1.2 Statement of the Problem

Fish is the most important single source of protein providing 16% of the animal

protein consumed by the world’s population (FAO, 1997). It is estimated that about

one billion people world- wide rely on fish as their primary source of animal protein

(FAO, 2000). The emergence of diseases associated with beef and poultry such as the Rift

Valley Fever, mad cow disease for beef and bird flu for poultry have all contributed to the

increase in consumption of fish. There is increased demand for fish in Kenya which cannot be

3
met by the capture fisheries with stocks stagnating due to overexploitation. The alternative has

been the growth in aquaculture with many people turning to farmed fish which is believed to

be of better quality whereas it is documented that aquatic environments harbor many bacteria

(Novotny, 2004). The use of organic waste for the fertilization of ponds is also a source of

pathogenic organisms that may be transmitted to humans via products of aquaculture. There is

little information on the quality of fish from Sagana fish farm and Masinga dam as far as the

bacterial microorganisms is concerned. There was therefore a need for this study to bridge this

gap.

1.3 Justification of the study

A lot of resources and international support are directed to ensure fish safety and

quality of fish for export, from the capture fisheries while aquaculture fish has received

very little or no attention. In Kenya a lot of emphasizes has been on the capture fisheries due

to the implications on the world trade while the aquaculture fish has received very little if any

attention regarding the bacterial flora, water quality and the type of feed used. This is mainly

due to the perception that aquaculture fish is assumed to be free of any contamination.

Information on the prevalence of bacterial pathogens that may be present in the

aquaculture industry in Kenya is unavailable. Additionally there is no data on the

status of sensitivity of these bacteria to anti-microbial agents used in the livestock

and horticulture industry in the country.

Against the above background, if fish is going to play a major role in both providing the

much needed protein and contributing to the national economy current information on such

aspect as fish borne diseases is required. And if aquaculture will reduce the gap

between supply and demand for fish and fishery products then there is need to establish the

bacterial flora of farmed fish, pond water and of sediment in the ponds and dams. This

4
study was carried out to identify the bacterial flora in the farmed fish and fish in the

dams and their anti-microbial response. The findings of this research will be useful in

managing aquaculture farms, formulating feeds for aquaculture fish and future choice of

antibiotics to use in aquaculture systems.

1.4 Research Questions

1. What is the bacteria flora of fish at Sagana fish ponds and Masinga dam in dry and wet

season?

2. Is there any significant difference of bacterial flora of fish between Sagana ponds and

Masinga dam, in dry season and wet season?

3. What is the anti microbial response of the micro-organisms from the fish at both

sources?

1.5 Objectives of the Study

1.5.1 Main Objective

To determine the bacterial flora of Tilapia, Catfish, water and water sediments and their

anti-microbial response from earthen ponds at Sagana fish farm and Masinga dam during

dry and wet seasons.

1.5.2 Specific Objectives

i. To isolate and identify bacterial flora species during dry and wet seasons at

Sagana fish ponds and Masinga dam

ii. To identify whether there is significant difference on the number of bacteria

flora species isolated in fish specimen types from ponds and dams, during dry

and wet season

iii. To determine anti- microbial response of the pathogenic bacteria isolates from

Sagana fish farm and the Masinga dams.

5
 CHAPTER TWO

LITERATURE REVIEW

2.1 Nutritional and economic value of fish

Fish is a vital source of food for people. It is man's most important single source of

high-quality protein, providing -16 % of the animal protein consumed by the world's

population (FAO, 1997). Fish oils are the only concentrated source of eicosapentanoic

acid (EPA) and docosahexaenoic acid (DHA). These fatty acids play a major role in the

development and maintenance of brain, and prevention of different pathologies mainly

the cardiovascular diseases and also psychiatric disorders such as stress, depression and

dementia (Bourne, 2005). It is estimated that about one billion people world- wide rely on

fish as their primary source of animal protein (FAO, 2000). FAO estimates the value of

fish traded internationally to be US $ 51 billion per annum. Over 36 milion people are

employed directly through fishing and aquaculture (FAO, 2000) and as many as 200

million people derive direct and indirect income from fish (Garcia and Newton, 1997).

In Kenya fresh water fish are mainly from Lake Victoria, rivers and the dams. Although

aquaculture in Kenya is not fully developed the potential is high and currently the annual

production is about 21,487metric tons valued at Kshs. 4, 633, 634, 000 (Fisheries

Department, 2012). The fisheries sector provides employment and income to over

500,000 Kenyans engaged in fish production, fish trade, industrial fish processing and

related enterprises (Fisheries Department, 2001).

2.2 Bacteria associated with farmed fish

Integrated fish farming combines livestock production with fish farming. In these arrangements,

animal manure is shed directly into a fish pond as fertilizer and supports the growth of

6
photosynthetic organisms. Several bacteria are reported to cause infection and

mortality in both fish and humans (Novotny, 2004) and these represent a particular

hazard caused either by handling infected fish on fish farms or in grocery stores or by

ingestion of raw or inadequately processed infected fish and /or contaminated fish

products. Bacterial pathogens are a major cause of infectious diseases and mortality

in wild fish stocks and fish reared in confined conditions. Disease problems

constitute the largest single cause of economic losses in aquaculture. Concurrent with

the rapid growth and intensification of aquaculture, increased use of water bodies,

pollution, globalization, and trans-boundary movement of aquatic fauna, the list of

new pathogenic bacterial species isolated from fish has been steadily increasing

(Ponnerassery et al., 2012).

The level of contamination of aquaculture products by the pathogenic bacteria will depend

on the environment and the bacteriological quality of the water in which the fish is cultured.

Tilapia, native to Africa and the Middle East is the second most common farm-raised food

fish in the world (Fitzsimmons, 1997). Aquatic animals take a large number of bacteria into

their gut and gills from water, sediment and food. The intestinal microflora may be

significant in fish spoilage (Kaneko, 1971) and may be involved in the spread of fecal

contaminants (Geldreich and Clarke, 1966). The microbial populations within the digestive

tract of fish are rather dense, the number of micro-organism being much higher than in the

surrounding water indicating that the digestive tract provides a favourable ecological niche

for these organisms (Horsley, 1977). Some normal bacterial microflora of water, such as

Pseudomonas fluorescens, Aeromonas hydrophilla, Edwardsiella tarda, Vibrio species and

Myxobacteria (Sugita et al., 1985), These can be found on the body surface or in the

intestinal tracts of fish under normal conditions but due to environmental stress may produce

7
epizootics diseases. There are two broad groups of bacteria that will contaminate fish. The

first group is the indigeneous microflora which occurs naturally in the environment such as

Aeromonas hydrophilla, Vibrio parahaemolyticus, Vibrio cholera, Vibrio vulnificus and

Llisteria monocytogenes. The other group is the non- indigenous bacteria that include the

members of the family Enterobacteriaceae such as Salmonella species, Shigella species and

Escherichia coli. A number of pathogenic microorganisms including Aeromonas,

Pseudomonas, Edwardsiella and streptococcus have been implicated in bacterial epidemics in

Tilapia (Oreochromis species) cultures (Al – Harbi, 1994; Al – Harbi and Uddin, 2003). The

frequency of contamination of fishery products by pathogenic microorganisms has been

considered a health hazard to consumers (Ingham and Potter, 1991).

2.2.1 Vibrio paraheamotyticus and other vibrios

Outbreaks of diarrhoea by Vibrio paraheamolyticus have been demonstrated in Japan and

Taiwan after ingestion of under cooked fish and raw products, sashimi and sushi

(Vuddhakul et al., 2000). Vibrio parahaemolyticus has been isolated from sea and estuary

waters on all continents with elevated sea water temperatures. It is isolated from fish

throughout the year in tropical climates. It causes acute gastroenteritis that is self

limiting, however, several cases require hospitalization and on rare occasions septicaemia

may occur. In the 1970s V. parahaemolyticus was the cause of 14 outbreaks of

gastroenteritis in USA (Barker et al., 1974), most of which occurred during the warmer

months and were attributed to seafood. Cholera is a highly contagious disease caused by

infection of the small intestines with Vibrio cholerae Ol and O139. It is characterized by

massive acute diarrhoea, vomiting and dehydration. It is often transmitted by water but

fish or fish products that have been in contact with contaminated water or faeces from

infected persons also frequently serve as a source of infection (Kam et al., 1995). In

Kenya the European Union banned fish imports from Kenya in 1996 citing poor

8
sanitary conditions at the beaches and lack of refrigeration /icing facilities (Fisheries

Dept, 1999).

2.2.2 Escherichia coli

Escherichia coli are enteric bacteria causing gastroenteritis. This bacteria together

with other coliforms and bacteria such as Staphylococcus spp. and sometimes

Enterococci are commonly used as indices of hazardous conditions during processing

of fish. Such organisms should not be present on freshly-caught fish (Chattopadhyay,

2000). An outbreak of diarrhoeal illnesses caused by ingestion of food contaminated

with enterotoxigenic E. coli was described in Japan (Mitsuda et al., 1998) associated

with eating Tuna paste. An outbreak caused by salted salmon roe contaminated,

probably during the production process, with enterohaemorrhagic E. coli (EHEC)

O157 occurred in Japan in 1998 (Asai et al., 1999). The salmon roe was stored

frozen for nine months but it appears that Enterohaemorrhagic E. coli (EHEC) O157

could survive freezing and a high concentration of NaCI and retained its

pathogenicity for humans. The isolation of E. coli in fishes grown in sewage-fed

farms and also in retail market fishes of Kolkata indicated contamination of fishes

with faecal matter of animal and human origin (Manna et al., 2008). Food products

that show evidence of faecal contamination are generally regarded as a greater risk to

human health, as they are likely to contain human-specific enteric pathogens. Some

strains of E. coli are capable of causing food-borne disease, ranging from mild

enteritis to serious illness and death (FAO/NACA/WHO, 1997).

2.2.3 Salmonella spp

Fish and shellfish are passive carriers of salmonella, which demonstrate no clinical disease

and can excrete Salmonella species without apparent trouble. Fish may therefore serve as a

9
vector for Salmonella species. In a Canadian outbreak of Salmonella enterica serotype

Paratyphi B was linked to aquariums (Gaulin et al., 2002). Another outbreak caused by drug

resistance Salmonella enterica subspecies serotype typhimurium DT104 was described

in Singapore (Ling et al., 2002). Dried anchovy was found to be the cause of infection.

Although most Salmonella outbreaks have been linked to poultry, the Hawaii

Department of Health studied 35 cases of Salmonella that arose from October 2007

to February 2008 and found that 86% of these patients had consumed raw fish in the

7 days before they got sick. In most cases, ahi, which is often made from imported

frozen tuna, was the reported fish consumed. In April 2008, eight of the nine cases of

Salmonella infection reported in the mainland United States also involved

consumption of raw tuna.

2.2.4 Staphylococcus aureus

Enterotoxins produced by Staphylococcus aureus are another serious cause of gastroenteritis

after consumption of fish and related products. In southern Brazil, Staphylococcus

aureus was isolated from 20% of 175 examined samples of fresh fish and fish fillets.

It was also detected during the process of drying and subsequent smoking of eels in Alaska in

1993 (Eklund et al., 2004).

2.2.5 Listeria monocytogenes

It is widely distributed in the general environment including fresh water, coastal water and in fish

from these areas. Contamination and recontamination may also take place during processing

(Huss et al., 2000). It is a psychotropic pathogen with the ability to grow at refrigerator

temperatures. An outbreak of listeriosis due to vacuum packed gravad and cold smoked

fish was described in at least eight human cases for eleven months in Sweden (Tham et al.,

2000).

10
2.2.6 Clostridium botulinum

The main habitat of C. botulinum is soil but it is also found in sewage, rivers, lakes, sea water,

fresh meat and fish (Haagsma, 1991). Clostridium botulinum type F caused deaths after

consumption of bought herrings without previous heating. Botulism caused by Clostridium

botulinum type B after eating fish salad was described by (Weber et al., 1993). Clostridium

botulinum type B which is found in marine and Lake Sediments and in fish intestines does not

grow or produce toxins in living fish but is carried passively. The bacterium becomes a hazard

when processing practices are insufficient to eliminate botulinal spores from raw fish.

2.2.7 Pseudomomas aeruginosa

They are widely found in water and are increasingly recognized as an emerging opportunistic

pathogen of clinical relevance. They have an ability to metabolize a variety of diverse nutrients and

to form biofilms and hence to survive in a variety of unexpected places. Pseudomonas

aeruginosa is one of the leading causes of nosocomial infections. The bacteria is

intrinsically resistant to many antimicrobial agents, including most β-lactams, the

older quinolones, chloramphenicol, tetracycline, macrolides, trimethoprim–

sulfamethoxazole and rifampin but a few strains are sensitive to drugs like the

ciprofloxacin (Rossolini and Mantengoli, 2005). This state of multi-drug resistance is

attributable to a concerted action of multidrug efflux pumps with chromosomally

encoded antibiotic resistance genes and the low permeability of the bacterial cellular

envelops. Besides intrinsic resistance it easily develops acquired resistance either by

mutation in chromosomally encoded genes or by the horizontal gene transfer of

antibiotic resistance determinants (Adelaide et al., 2009).

2.2.8 Aeromonas species

This bacterium can also be found in fresh, salt, marine estuarine, chlorinated water. It

11
can survive in aerobic and anaerobic environments. It is very resistant to chorine,

refrigeration or cold temperatures making it hard to kill and posing a danger to fish

processing. It occurs in contaminated environments and is also ingested through food

products that have been infected with the bacterium (Daskalov, 2006). It causes

gastroenteritis which occurs mostly in young children and people who have

comprised immune systems or growth problems. About 8.1% of cases of acute

enteric diseases in 458 patients in Russia were caused by Aeromonas species

(Pogorelova et al., 1995). This could increase the hazard of food contamination,

particularly where there is a possibility of cross- contamination with ready-to-eat

food products. Some strains are important fish pathogens in aquaculture (Pillay,

1990), while others have been implicated in food borne disease (Morgan and Wood,

1988). Aeromonads can be causative agents not only of human enteritis

(Sukroongreung et al., 1983 ), but also of a fatal septicemia as recorded in a 15- year

old healthy girl, where the causative agent was found to be Aeromonas sobria

(Shiina et al., 2004).

2.2.9 Citrobacter freundii

Citrobacter species are found in water, soil and decaying matter and can be isolated

from the faeces of man and animals. They are small, Gram-negative, non spore

forming rods, and belong to the family Enterobacteriaceae. Citrobacter species

grows best at moderate temperatures but can also grow at low temperatures (7 ºC).

Citrobacter is one of the major genera of bacteria that are found on fresh meat,

minced meat, poultry, plants and plant products (Kleeberger and Busse, 1975; Jay,

2000). Sources of these food contaminants may be the original environment (such as

water and soil) of fish, meats and vegetables. Treated wastewater, for example, is

reused for irrigation and other purposes in many countries (WHO, 1989). Citrobacter

12
is one of the prevalent species in the influent and effluent of wastewater treatment

plants (Abu-Ghazaleh, 2001) therefore; vegetables, fish and other foods in contact

with this water may be contaminated. It has strains that have inducible ampC genes

encoding resistance to ampicillin. The first generation cephalosporins resistance

encountered in this organism could also be as a result of plasmid- encoded resistance

genes (Abu-Ghazaleh, 2001).

2.2.10 Edwardsiella tarda

This is a member of the family Enterobacteriaceae. Edwardsville spp. has been

implicated in gastroenteritis in humans and in bacteremic infections that include

wound abscesses and meningitis (Sakazaki et.al., 1971). It has been isolated from a

diseased pig, an ostrich and was also implicated as the causative agent of a disease in

pond-reared eels (Wakabayashi and Egusa, 1973). Incidence of E. tarda in fishes

from freshwater aquaculture environment and retail market have been reported

(Pankajkumar, 2009), and human liver abscess caused by E. tarda bio group in India

(Manchanda et al., 2006) have also been reported.

2.3 The Analytical Profile Index (API 20E)

The Analytical Profile Index (API) is a miniaturized panel of biochemical tests

compiled for identification of groups of closely related bacteria. Different test panels

are prepared in dehydrated forms which are reconstituted upon use by addition of

bacterial suspensions. After incubation, positive test results are scored as a seven-

digit number (profile). Identity of the bacterium is then easily derived from the

database with the relevant cumulative profile code book or software.

The API 20E used was biochemical panel for identification and differentiation of

13
members of the family Enterobacteriaceae. In API 20E for identification of members

of the family Enterobacteriaceae, the plastic strip holds twenty mini-test chambers

containing dehydrated media having chemically-defined compositions for each test.

2.4 Antimicrobial Resistance

The wide application of antibiotics by humans has led to large-scale dissemination of

bacteria resistant to antibiotics in water basins (Schwartz et al., 2003; Dang et al,.

2006). For antibiotic resistance to develop, it is necessary that two key elements

combine: the presence of an antibiotic capable of inhabiting the majority of bacteria

present in a colony and heterogeneous colony of bacteria where at least one of this

bacterium carries the genetic determinant capable of expressing resistance to the

antibiotic (Levy and Marshall, 2004). Resistance to antibiotics can be natural or

acquired and can be transmitted horizontally or vertically. Whereas the natural form

of antibiotic resistance is caused by a spontaneous gene mutation in the lack of

selective pressure due to presence of antibiotics and is far much less common than

the acquired one, it can also play a role in the development of resistance (Alanis,

2005). Sagana fish farm practices integrated fish farming which involves confined

units with little exchange of water. Manure from livestock production is administered

to fish ponds. It has been shown that such practice supports the growth of mainly

photosynthetic organisms (Little and Edwards, 1999). This integrated fish farming

system produces high yields with low input, with the fish receiving limited, if any,

supplementary feed. In contrast, the livestock on the integrated farms, which includes

chickens, is reared intensively, and antimicrobial agents are used for prophylactic

and therapeutic treatment. Within integrated fish farming systems, antimicrobials,

their residues, and antimicrobial-resistant bacteria may enter the fish ponds through

14
animal manure and/or excess feeding and are potential sources of antimicrobial-

resistant bacteria.

Antimicrobial resistance in traditional fish farming systems in temperate waters has

been intensively studied (Alderman and Hastings, 1998). A high incidence of

bacteria resistant to the antimicrobials used in aquaculture, including multiple

resistant bacteria, has been found in fish farms and the surrounding aquatic

environments. Residues of antimicrobials have been found in the sediments of

marine fish farms (Jacobsen and Berglind, 1988; Björklund et al., 1990).

Accumulation of surplus antimicrobials and antimicrobial residues may occur in

integrated fish farms when the ponds are only rarely emptied at the time of fish

harvest. Such a build up could establish selective pressure favoring selection and

growth of antimicrobial-resistant bacteria. Although increased levels of antimicrobial

resistance in and around fish farms may only occur transiently, there is a potential

risk that antimicrobial resistance genes could be disseminated into a wide range of

aquatic environmental bacteria. However, resistance to one antimicrobial within a

class of antimicrobials often confers resistance to other members of the same group

(cross-resistance). Potential transfer of resistant bacteria and resistance genes from

aquaculture environments to humans may occur through direct consumption of

antimicrobial-resistant bacteria present in fish and associated products.

2.5 Histamine fish poisoning

Histamine poisoning is one of the most significant causes of illness associated with seafood.

It is formed in the fish post mortem by bacteria decarboxylation of the amino acid histidine. The

histamine producing bacteria are certain Enterobacteriaceae, some Vibrio spp few

Clostridium and Lactobacillus sp. The most potent are Morganella morganii, Klebsiella

15
pneumoniae and Hqfhia alvei (Stratten and Taylor, 1991). Some are present in the normal

micro flora of live fish others are derived from post-catching contamination on fishing

vessels, at processing plant or in the distribution system. Once produced in the fish the

risk of provoking disease is very high since it is heat resistance. Histamine formation can be

prevented by rapid cooling of fish after catching and adequate refrigeration during handling

and storage. A total of 22 cases of histamine fish poisoning after the consumption of tuna

burgers, tuna salad and fillets were reported in North Carolina from 1998 to 1999 (Barker

et al., 1974). However, the human body will tolerate a certain amount of histamine

without any reaction. The ingested histamine will be detoxified in the intestinal tract by at

least 2 enzymes, the diamine oxidase (DAO) and Histamine N-methyltransferase (HMT)

(Taylor, 1986). This mechanism can be prevented if the intake of histamine and other

biogenic amines such as cadaverine and putrescine is very high. However, the impact of

these infections on children, the aging population and the imuno-compromised, cannot

be under estimated. The ease of world shipment of fresh and frozen food and the

development of new food industries including aquaculture only compounds the problem

(Todd, 1997).

2.6 Factors contributing to fish contamination

It is well known that microbiological activity is greatly influenced by temperature

changes. In the temperature range from 0°C to 25° C microbiological activity is of great

importance. Many bacteria are unable to grow at temperatures below 10°C and even

psychrotrophic organisms grow very slowly and sometimes with extended lag phases.

When temperatures approach 0° C the growth rate is less than one tenth of the rate at the

optimum growth temperature (FAO, 1995). The micro flora responsible for spoilage of fresh fish

change with changes in storage temperature. At low temperatures (0°-5°C), Schewanella

putrefaciens, Photobacterium phosporeum, Aeromonas spp and Pseudomonas spp cause

16
spoilage (Huss, 1994). At high storage temperatures (15° -30°C) different species of

Vibrionaceae, Enterobacteriaceae and Gram- positive organisms are responsible for spoilage

(Gram et al., 1990).

The ponds and rivers that harbour the fish may be the source of contaminants 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 from contaminated 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 (Doyle and Ericson, 2006).

Another factor that affects the microbial load on fish is the accumulation of waste feeds in

ponds which stimulates the growth of bacteria including human pathogens which can

contaminate products and lead to food-borne diseases. The use of artificial feeds

supplemented with antibiotics in the feeds could lead to residues remaining in the fish

which in turn will lead to the development of antibiotic-resistant bacteria in the food chain

(Doyle and Ericson, 2006). The quality and storage life of many fish decreases once the

fish is gutted because it exposes the fish belly and cut surfaces to the air rendering them most

susceptible to oxidation and decolourization. Oxidation takes place in the lipid fraction of the

fish and involves oxygen and the unsaturated lipid. It is formation of hydrogen peroxides

which is degraded to form aldehydes and ketones. It is initiated and accelerated by heat,

light and several organic and inorganic substances (Doyle and Ericson, 2006).

17
 CHAPTER THREE

MATERIALS AND METHODS

3.1 Study Site

The study was carried out at Masinga Dam and Sagana Aquaculture Centre situated

on Tana –river and Kirinyaga districts respectively. Masinga dam (Figure 3.1) is

situated on Tana River, the main catches are Tilapia and Catfish. Dam fisheries

account for about the Kshs. 34million per year with most consumption being in

Nairobi City. Sagana Aquaculture Centre,( Figure 3.1) is a Department of Fisheries

breeding farm in Sagana town, Kirinyaga District (100 km North East of Nairobi,

altitude 1230 m, latitude 0°39'S and longitude 37°12'E). It covers an area of

approximately 50 hectares of which 18 hectares are covered by ponds; on average

each pond covers an area of about 40 by20M2 with a depth of 1meter. Water is

diverted from river Ragati and delivered by gravity through a canal. It is one of the

two main national fish hatcheries of the fisheries department and acts as a training

centre for fish farmers in aquaculture.

The farm is involved in the culture of Nile Tilapia and African Catfish as the main

species among others. The farm provides quality fish feeds to farmers and

demonstrates economic viability of integrated fish farming and it also conducts

research. Fish farming is carried out on still- water earthen ponds under semi

intensive systems. The ponds are fertilized using artificial fertilizers and organic

manure from cattle, chicken and kitchen wastes to enhance growth for the

phytoplankton. The other alternative feeds used are rice bran, wheat germ, maize

bran. The experimental period started during the dry season in August 2007 and

continued to the rainy season of December 2007.

18
 Masinga dam
Sagana fish
farm

Figure 3.1: Map of Kenya showing Sagana fish farm and Masinga dam

3.2 Sample size

Sample size was determined using the ICMSF sampling standard which relates the

stringency of the sampling plan to the degree of hazard of the food. A 3-class plan is

used when the health hazard is low. In this plan n = 5 and c= 3, n is the number of

samples drawn and c is the maximum allowable number of positive results.

19
3.3 Study design
The study design adopted was purposive study.

3.4 Sampling procedure

3.4.1 Collection of fish samples

Fish was sampled from 20 organically fertilized ponds at the Sagana fish farm the

ponds were selected at interval of 4 ponds and 5 pieces of table size Tilapia were

harvested using a scoop net and taken to the laboratory in a cool box. Once in the

laboratory; the fish were aseptically skinned to get the skin sample. Up to 25g of fish

skin was weighed and mixed with 225ml of buffered peptone water in a sterile

blender. It was blend and a portion of the pure mix inoculated into the MacConkey

agar culture media. In order to have distinguishable colonies, diluted subcultures

were made from the initial culture after overnight incubation at 37 0 C at dilutions of

1:10, 1:100 and 1: 1000 using peptone water and incubated overnight 370C. Growth

of distinct colonies was achieved at the 1: 100 dilutions. The colonies were sub-

cultured in different selective media Hektoen enteric (HE) agar, xylose-lysine

deoxycholate (XLD) agar and Salmonella-Shigella (SS) agar and incubated. The

colonies were morphologically identified and then subjected to biochemical tests for

further identification. Final identification was done using API 20E method.

For the gut sample the fish was cleaned using 70% alcohol and an incision was

made over peritoneal cavity and the fish dissected to get the gut contents. The gut

contents were combined and weighed as was done for the skin and the same

procedure was followed and again growth of distinct colonies was at 1: 100 dilutions.

The same procedure was carried out for the dam fish during the two seasons.

20
3.4.2 Collection of Water samples

Water was collected from a depth of about 20cm beneath the surface in sterile bottles

of 100ml at one end of the pond and at the center of the pond. The water sample was

enriched using peptone water and incubated at 370C overnight this was inoculated to

MacConkey culture media and incubated at 370C overnight. Growth of distinct

colonies was obtained at the 1: 10 dilution. They were sub cultured in selective

media and incubated. The colonies were morphologically identified and further

subjected to biochemical tests for further identification. The final identification was

by use of API 20E method.

Dam water was collected in the early hours of morning and taken to the laboratory

within 2 hours. The sample was collected using sterile bottles away from the bank at

a depth of one foot below the surface and the mouth directed towards the current and

then it was filled and covered immediately. It was stored in a cooler box during

transportation. The same procedure used for the pond water was applied to the dam

water.

3.4.3 Collection of water sediments

Sediments samples were collected from the bottom of the pond and dam using a

Ekman grab in all the ponds. The sediments were collected at the edge and at the

center of the ponds. The sediments were then taken to the Laboratory where they

were mixed and enriched using peptone water cultured in MacConkey and incubated

at 37 0C overnight. The colonies were sub-cultured in selective media. The colonies

were morphologically identified and then subjected to the biochemical tests. Final

identification was done using API 20E method.

21
3.5 The Analytical Profile Index (API 20E)

All test chambers were rehydrated by inoculation with a saline suspension of a pure

culture of the bacterial strain subjected to identification. After incubation in a

humidity chamber for 18 to 24 hours at 37°C, the color reactions were read. The

results of the test reactions were converted to a seven-digit code. The code was then

looked up in the database book for the genus and species identification of the test

microorganism.

3.6 Antibiotic susceptibility testing

3.6.1 Antibiotic sensitivity profile

The antibiotic sensitivity testing was performed on all the isolates against commonly

used antimicrobial agents’ (Table 3.1). Kirby – Bauer disk diffusion test was used as

per the recommended standard of the National Committee for Clinical Laboratory

Standards (NCCLS, 2009). After incubation at 370C for 24 hours the diameters of

zones of inhibition were measured and compared with control organism E. coli the

ATCC 25922.

22
Table 3.1: Antimicrobial agents used for sensitivity testing

No. Antimicrobial agent Concentration

1 Ampicillin (Amp) 10µg/ml

2 Chlorampenicol (C) 30µg/ml

3 Streptomycin (S) 10µg/ml

4 Tetracycline (Te) 30µg/ml

5 Nalidixic acid (Na) 30µg/ml

6 Ciprofloxacin (Cip) 5µg/ml

7 Gentamycin (Gen) 10µg/ml

8 Cefuroxine (Cxm) 30µg/ml

9 Amoxicillin (Aml) 5µg/ml

10 Cefotaxine (CTX) 30µg/ml

23
 CHAPTER FOUR

RESULTS

4.1 Bacteria isolates per specimen type

A total of 91 bacteria were isolated from six specimen types namely; Tilapia Gut,

Tilapia skin, Catfish skin, Catfish Gut, water and water sediments. Tilapia gut had

the highest proportion (20; 22%) of bacteria isolates compared to the rest. Tilapia

skin and catfish skin each had 16 (17.6%) of bacteria isolated. Catfish gut and water

had the same number of bacteria isolates 15 (16.5%). Water sediments had the lowest

proportion (9; 9.9%) of bacteria isolated compared to the rest of specimens (Figure

4.1).

Figure 4.1: Bacteria isolates from various collected samples

4.1.2 Types of bacteria isolates from Tilapia, Catfish, Water, Water sediments.

A total number of 91 (100%) bacterial isolates were indentified of which Citrobacter

freundii were 16 (17.6%) compared to the rest of bacteria isolated. Citrobacter

freundii and E.coli are the only bacteria that occurred in five specimen type except in

water sediments and Tilapia skin respectively (Table 4.1).

24
In tilapia skin the bacterial isolates were 16 (17.6%) same as the bacterial isolates in

Catfish skin. The bacteria isolates that occurred both in Tilapia Skin and Catfish skin

were: Aeromonas sobia, Citrobacter freundii, E.coli, Edwardsiella tarda,

Enterobacter cloacae, Enterobacter sakazakii, Proteus mirabilis and Pseudomonas

aeruginosa (Table 4.1).

In Tilapia Gut a total number of 20 (22.0%) bacterial isolates were identified. On the

other hand, 15 (16.5%) of bacteria were isolated from Catfish Gut. Bacterial isolates

that occurred in both Tilapia gut and Catfish skin were; Aeromonas sobia,

Citrobacter freundii, E.coli, Edwardsiella tarda, Enterobacter sakazakii, .and

Pseudomonas fluorescenes (Table 4.1)

In water, 15 (16.5%) bacteria were isolated compared 9 (9.9%) bacterial isolates

from water sediments. Bacterial isolates that occurred both in water and water

sediments were; E.coli, Enterobacter fergusonii, Pseudomonas fluorescenes and

Salmonellaspp.(.(Table4.1)

25
Table 4.1: Bacteria isolates in Tilapia, Catfish, Water and Water sediments
Isolates Tilapia Skin Catfish Skin Tilapia Gut Catfish Gut Water Water sediments Total
Acinetobacter spp - - - - 1 - 1 (1.1%)
Aeromonas sobia 2 2 1 1 - - 6 (6.6%)
Chromobacterium violaceum - - 1 - - - 1 (1.1%)
Citrobacter freundii 4 2 5 3 2 - 16 (17.6%)
E.coli - 1 2 2 5 4 14 (15.4%)
Edwardsiella tarda 1 1 2 2 - - 6 (6.6%)
Enterobacter agglomerans - 1 - - - 1 2 (2.2%)
Enterobacter amnigenus - - 1 - - - 1 (1.1%)
Enterobacter cloacae 1 1 - - 1 - 3 (3.3%)
Enterobacter fergusonii - - - - 1 1 2 (2.2%)
Enterobacter sakazakii 1 1 1 3 - - 6 (6.6%)
Klebsiella onithnolytica 1 - - - - - 1 (1.1%)
Klebsiella pneumonia 1 - 1 - - - 2 (2.2%)
Plesiomonas shigelloides - - - - 1 - 1 (1.1%)
Proteus mirabilis 1 1 - - - 1 3(3.3%)
Providentia stuartii - 3 - 2 - - 5 (5.5%)
Pseudomonas aeruginosa 2 3 3 - 1 - 9 (9.9%)
Pseudomonas fluorescenes - - 1 1 1 1 4 (4.4%)
Salmonella spp 1 - 1 - 1 1 4 (4.4%)
Shigella boydii - - - 1 - - 1 (1.1%)
Vibrio mechnikovii 1 - - - 1 - 2 (2.2%)
Vibrio vulnificus - - 1 - - - 1 (1.1%)
Total 16 (17.6%) 16 (17.6%) 20 (22.0%) 15 (16.5%) 15 (16.5%) 9 (9.9%) 91 (100.0%)

26
4.2 Types of bacteria isolates from Sagana pond and Masinga dam
The bacteria isolates from sagan ponds were 54 (59.3%) while isolates from Masinga

dam were 37 (40.7%) Citrobacter freundii were 16 (17.6%) of which 10 (11.0%)

were isolated from Sagana pond while 6 (6.6%) were isolated from Masinga dam. In

all types of bacteria isolated, Aeromonas sobia, Citrobacter freundii, Ecoli,

Edwardsiella tarda, Proteus mirabilis, Pseudomonas aeruginosa and Pseudomonas

fluorescenes occurred in both Sagana pond and Masinga dam. (Table 4.2)

Table 4.2: Types of bacteria isolated from Sagana pond and Masinga dam

Bacteria isolated Sagana Masinga Sagana Masinga Total

Pond dam Pond dam
n (%) n (%) n (%)
Acinetobacter spp + - 1 (1.1) 0 (0.0) 1 (1.1)
Aeromonas sobia* + + 4 (4.4) 2 (2.2) 6 (6.6)
Chromobacterium + - 1(1.1) 0 (0.0) 1 (1.1)
violaceum
Citrobacter freundii* + + 10 (11.0) 6 (6.6) 16 (17.6)
Ecoli* + + 6 (6.6) 8 (8.8) 14 (15.4)
Edwardsiella tarda* + + 4 (4.4) 2 (2.2) 6 (6.6)
Enterobacter aggromerans - + 0 (0.0) 2 (2.2) 2 (2.2)
Enterobacter amnigenus + - 1(1.1) 0 (0.0) 1 (1.1)
Enterobacter fergusoni + - 2 (2.2) 0 (0.0) 2 (2.2)
Enterobacter sakazakii - + 0 (0.0) 6 (6.6) 6 (6.6)
Enterobacter cloace - + 0 (0.0) 3 (3.3) 3 (3.3)
Klebsiella onithnolytica + - 1 (1.1) 0 (0.0) 1 (1.1)
Klebsiella pneumonia + - 2 (2.2) 0 (0.0) 2 (2.2)
Plesiomonas shigelloides - + 0 (0.0) 1 (1.1) 1 (1.1)
Proteus mirabilis* + + 1 (1.1) 2 (2.2) 3 (3.3)
Providencia stuartii + - 5 (5.5) 0 (0.0) 5 (5.5)
Pseudomonas aeruginosa* + + 7 (7.7) 2 (2.2) 9 (9.9)
Pseudomonas fluorescenes* + + 2 (2.2) 2 (2.2) 4 (4.4)
Salmonella spp + - 4 (4.4) 0 (0.0) 4 (4.4)
Shigella boydii + - 1 (1.1) 0 (0.0) 1 (1.1)
Vibrio mechnikovii + - 2 (2.2) 0 (0.0) 2 (2.2)
Vibrio vulnificus - + 0 (0.0) 1 (1.1) 1 (1.1)
Total 54 (59.3) 37 (40.7) 91(100)
+ /- Species occurred/ did not occur, * Bacteria isolated both in Sagana and Masinga
dam

27
4.2.1 Bacteria isolates from Sagana pond and Masinga dam by specimen type

Tilapia gut had a total of 20 (22.0%) bacteria isolates of which 12 (13.2%) were from

Sagana ponds and 8 (8.8%) bacteria isolates were from Masinga. Water sediments

had the lowest proportion (9; 9.9%) of bacteria isolates both from Sagana ponds (4;

4.4%) and Masinga dam (5; 5.5%) respectively. When the results were subjected to

chi-square, there was no significant difference of bacteria flora species isolated

between pond and dam water  2 =3.853, df=5, =0.571) (Table 4.3).

Table 4.3: Number of bacteria isolated from Sagana farm and Masinga dam

Sagana Masinga
Farm dam Total P-
Specimen type n (%) n (%) n (%)  2
Df Value

Tilapia
Skin 12 (23.2%) 4 (4.4%) 16 (17.6%) 4.00 1 0.050
Tilapia Gut 12 (23.2%) 8 (8.8%) 20 (22.0%) 0.800 1 0.371
Cat fish Skin 9 (9.9%) 7 (7.7%) 16 (17.6%) 0.250 1 0.617
Catfish Gut 7 (7.7%) 8 (8.8%) 15(16.5%) 0.670 1 0.796
Water 10 (11.0%) 5 (5.5%) 15(16.5%) 1.667 1 0.197
Water
sediments 4 (4.4%) 5 (5.5%) 9(9.9%) 0.111 1 0.739
Total 54 (59.3%) 37 (40.7%) 91(100%) 3.853 5 0.571
*Significant at 0.05

4.3 Bacteria isolated from Tilapia, Catfish, Water and Water sediments
in dry and wet seasons

Bacteria isolates from tilapia gut were (20; 22%) of which 11 (12.1%) were isolated

during dry seasons while 9 (9.9%) of bacteria isolated during wet season. There were

more bacteria isolates during dry season as compared to wet season in all specimen

types except tilapia skin specimen where (6; 6.6%) bacteria were isolated in dry and

28
10 (11.0%) in wet season respectively. The reason adduced for higher number of

bacteria isolates during dry season compared to wet season according to Wemedo

(2002) points out that during the wet seasons, lower temperatures inhibited microbial

activity. Another reason given attributed to this phenomenon is that saturation of the

soil by rain, limits activity by reducing aeration (Marshall and Devinny, 1998).

(Figure 4.2)

Figure 4.2: Bacteria isolates in dry and wet season by specimen type

4.3.1 Types of bacteria isolated from the dry and wet season

During the dry season 50 (54.9%) bacteria were isolated while 41 (45.1%) bacteria

were isolated during the wet season. The number of Citrobacter freundii isolated

from both dry and wet season were 16 (17.6%) of which 9 (9.9%) were isolated

during dry season while 7 (7.7%) isolated during wet season. Citrobacter freundii,

Aeromonas sobia, Acinetobacter spp, Edwardsiella tarda, E.coli, Pseudomonas

aeuroginosa, Enterobacter cloacae, Enterobacter sakazakii and Providencia stuartii

were isolated in both seasons (Table 4.4).

29
Table 4.4: Bacteria isolates from dry and wet season

Bacteria isolates Dry Wet Dry Season Wet Season Total

Season Season n (%) n (%) n (%)

Acinetobacter spp* + + 1 (1.1) 1 (1.1) 1 (1.1)

Aeromonas sobia* + + 4 (4.4) 2 (2.2) 6 (6.6)
Chromobacterium - + 0 (0.0) 1 (1.1) 1 (1.1)
violaceum
Citrobacter freundii* + + 9 (9.9) 7 (7.7) 16 (17.6)
Ecoli* + + 6 (6.6) 8 (8.8) 14 (15.4)
Edwardsiella tarda* + + 3 (3.3) 3 (3.3) 6 (6.6)
Enterobacter + - 2 (2.2) 0 (0.0) 2 (2.2)
aggromerans
Enterobacter amnigenus + - 1 (1.1) 0 (0.0) 1 (1.1)
Enterobacter fergusoni + - 2 (2.2) 0 (0.0) 2 (2.2)
Enterobacter sakazakii* + + 4 (4.4) 2 (2.2) 6 (6.6)
Enterobacter cloace* + + 2 (2.2) 1 (1.1) 3 (3.3)
Klebsiella onithnolytica - + 0 (0.0) 1 (1.1) 1 (1.1)
Klebsiella pneumonia - + 0 (0.0) 2 (2.2) 2 (2.2)
Plesiomonas shigelloides + - 1 (1.1) 0 (0.0) 1 (1.1)
Proteus mirabilis - + 0 (0.0) 3 (3.3) 3 (3.3)
Providencia stuartii* + + 3 (3.3) 2 (2.2) 5 (5.5)
Pseudomonas aeruginosa* + + 4 (4.4) 5 (5.5) 9 (9.9)
Pseudomonas fluorescenes - + 0 (0.0) 4 (4.4) 4 (4.4)
Salmonella spp + - 4 (4.4) 0 (0.0) 4 (4.4)
Shigella boydii + - 1(1.1) 0 (0.0) 1(1.1)
Vibrio mechnikovii + - 2 (2.2) 0 (0.0) 2 (2.2)
Vibrio vulnificus + - 1(1.1) 0 (0.0) 1(1.1)
Total 50 (54.9) 41 (45.1) 91(100)
+ Species occurred, - Species did not occur, * Bacteria isolated in both dry and wet
season

4.3.2 Bacteria isolates during wet and dry season by specimen type

All bacterial isolates were 91 with tilapia fish gut had 20 (22.0%) of which 6 (6.6%)

were during dry season while 10 (11.0%) were during wet season. There was no

significance difference between the number of bacteria isolated from Tilapia fish gut

and the two seasons (p>0.05). This was same to other specimen types in spite of

more bacteria isolates during the dry season (Table 4.5) including Tilapia fish skin

which had 6 (6.6%) and 10 (11.0%) of bacteria isolated from both dry and wet

seasons respectively, There was no significance difference to show any association

30
of bacteria isolates and the source (  2 =3.006, df=5, P=0.699).

Table 4.5: Bacteria isolates in specimen type by season

Wet
Specimen type Dry Season Season Total 2 df P-value
Tilapia Skin 6 (6.6%) 10 (11.0%) 16(17.6%) 1.000 1 0.317
Tilapia Gut 11(12.1%) 9(9.9%) 20(22.0%) 0.200 1 0.655
Cat fish Skin 8 (8.8%) 7(7.7%) 15(16.5%) 0.067 1 0.796
Cat fish Gut 9 (9.9%) 6(6.6%) 15(16.5%) 0.600 1 0.439
Water 10 (11.0%) 6(6.6%) 16(17.6%) 1.000 1 0.317
Water Sediments 6 (6.6%) 3(3.3%) 9(9.9%) 1.000 1 0.317
Total 50 (54.9%) 41(45.05%) 91(100%) 3.006 5 0.699

31
4.4 Correlation between Bacteria isolates from Specimen types, Site and Season
There was a strong positive correlation between bacteria isolated during dry and wet

season from the specimen and the site (Ponds and Dams). The more bacteria isolates

from the two sites, the higher significance difference between the bacteria isolated

during dry season and wet season (r=0.734, P=0.000) (Table 4.6). Pearson’s

correlation did not indicate any significant correlation between the bacteria isolated

from specimen and the site. Correlation between the two variables was weak and

negatively correlated (r=0.734, P=0.000) (Table 4.6). On the other hand, there was a

weak positive correlation between the bacteria isolated from specimen types and the

site (Sagana ponds and Masinga dam) (r=0.136, P=0.197) (Table 4.6).

Table 4.6: correlation between specimen, site and season

Season Site

Pearson Correlation 0.734** 1

Site Sig. (2-tailed) 0.000

N 91 91

Specimen Pearson Correlation -0.162 0.136

Sig. (2-tailed) 0.124 0.197

N 91

**. Correlation is significant at the 0.01 level (2-tailed).

32
4.5 Overall antibacterial response of isolates
All bacteria isolates were examined for susceptibility to commonly used

antimicrobial agents. The zones of inhibition were read after incubation, compared

against a standard measurement and recorded as resistant, intermediate or sensitive.

CIP antibiotic was susceptible to all (100%) bacterial isolates while on the other hand

none of the bacterial isolates that registered resistant to CXT, GEN and CIP

antibiotics. The drug with the highest resistance was AML with 60 (65.9%) of

bacteria isolated registering resistance followed by AMP at 56 (61.5%) bacterial

isolates. On average 64 (70.3%) of bacteria isolated registered susceptibility, 18

(20%) registered resistance to drugs while 9 (9.6%) registered intermediate. (Table

4.7and Figure 4.3).

Table 4.7: Antimicrobial Response of Isolates to various Antibiotics

Resistant Sensitive Intermediate Total

Antibiotic N % n % n % N %
AML 60 65.9 27 29.6 4 5.5 91 100
CXT 0 0 83 91.2 8 7.14 91 100
CIP 0 0 91 100 0 0 91 100
S 4 4.4 69 75.8 18 19.7 91 100
AMP 56 61.5 29 31.8 6 6.6 91 100
GEN 0 0 90 98.9 1 1.1 91 100
TE 29 31.8 40 43.9 22 24.2 91 100
C 25 27.5 47 51.6 19 20.9 91 100
NA 4 4.4 78 85.7 9 9.8 91 100
CXM 4 4.4 86 94.5 1 1.1 91 100
Average 18 20.0 64.0 70.3 9 9.6 91 100

33
Figure 4.3: Percentage distribution of antimicrobial response of isolates to various
antibiotics

4.6 Antibacterial response of isolates from Sagana Ponds

The total isolates from the pond were 54 of which 38 (70.4%) registered resistance to

7 antimicrobial agents. Bacterial isolates from Sagana registered high resistance to

AML and AMP drugs, 38 (70%) and 36 (67%) respectively. The rest of isolates

resistance registered to antibiotics were as follows; 16 (30%) were resistant to TE, 15

(28%) to C and 5 (10%) to NA and S respectively. There was no resistance registered

to CXT, CIP and GEN for bacterial isolates from Sagana ponds. (Figure 4.4)

34
Figure 4.4: Number of Isolates resistant to antibiotics from Sagana

In regards to bacteria types isolated from Sagana pond, Salmonella spp showed

resistance to AML, AMP and CXM. Most of bacterial isolates from Sagana ponds

showed resistance to an average of 2 to 3 drugs with an exception of klebsiella

pneumonia which registered resistance to four antibiotics namely; AML, S, AMP,

TE and C. There was variation in antibacterial response of isolates from Sagana pond

(F=8.4, P=0.000) (Table 4.6).

35
Table 4.6 Antimicrobial Response of Isolates from Sagana

AML CTX CIP S AMP GEN TE C NA CXM

Isolates No. of isolates
R S I R S I R S I R S I R S I R S I R S I R S I R S I R S I
Citrobacter freundii 10 9 1 0 0 10 0 0 10 0 0 5 5 9 1 0 0 10 0 2 4 4 3 4 3 0 10 0 0 10 0
Aeromonas sobia 4 4 3 0 0 7 0 0 7 0 0 6 1 6 1 0 0 7 0 0 7 0 0 7 0 0 7 0 0 7 0
Vibrio mechnikovii 2 2 0 0 0 2 0 0 2 0 0 1 1 2 0 0 0 2 0 0 2 0 1 1 0 0 2 0 0 2 0
Salmonella spp 4 1 1 0 0 2 0 0 2 0 0 2 0 2 2 0 0 2 0 0 2 0 0 2 0 0 2 0 0 2 0
Edwardsiella tarda 4 0 1 1 0 2 0 0 2 0 0 2 0 0 1 1 0 2 0 1 1 0 1 1 0 0 2 0 0 2 0
Shigella boydii 1 1 0 0 0 1 0 0 1 0 0 1 0 1 0 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0
Ecoli 6 0 5 1 0 6 0 0 6 0 0 6 0 0 4 1 0 6 0 3 2 1 2 2 2 0 4 2 0 6 0
Enterobacter 1 1 0 0 0 1 0 0 1 0 0 1 0 1 0 0 0 1 0 0 1 0 0 0 1 0 1 0 0 1 0
amnigenus
Enterobacter 2 1 0 0 0 1 0 0 1 0 0 0 1 1 0 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0
fergusonii
Acinetobacter spp 1 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 1 1 1 0 0 1 0 0 1 0
Pseudomonas 7 7 0 0 0 6 1 0 7 0 1 4 2 5 1 1 0 6 1 3 0 4 5 0 2 4 3 0 3 4 0
aeruginosa
Klebsiella 1 1 0 0 0 1 0 0 1 0 0 1 0 1 0 0 0 1 0 1 0 0 0 0 0 0 1 0 0 1 0
onithnolytica
Chromobacterium 1 0 1 0 0 0 1 0 1 0 0 1 0 0 1 0 0 1 0 1 0 0 0 0 0 1 0 1 0 0 1
violaceum
Proteus mirabilis 1 2 0 0 0 2 0 0 2 0 0 2 0 1 0 1 0 2 0 0 1 1 1 1 0 0 2 0 0 2 0
Pseudomonas 2 1 0 0 0 2 0 0 2 0 0 1 2 0 2 0 0 0 2 0 0 2 0 0 1 1 0 2 0 1 1
Flurosecenes
Klebsiella pneumonia 2 2 2 0 0 4 0 0 4 0 1 3 0 2 2 0 0 4 0 2 1 1 1 3 0 0 4 0 0 4 0
Providencia stuartii 5 3 0 0 0 3 0 0 3 0 0 3 0 3 0 0 0 3 0 1 2 0 1 2 0 0 3 0 0 3 0
Total 54 34 15 2 0 52 2 0 54 0 2 40 11 34 16 4 0 51 2 14 26 14 16 26 9 6 44 5 3 48 2
% 100 63 28 4 0 97 4 0 100 0 4 74 20 63 30 7 0 94 4 26 48 26 30 48 17 11 82 9 6 89 4
F=8.4, df=127, P-value=0.000

36
4.7 Antibacterial Response of Isolates from Masinga
Bacterial isolates from Masinga dam registered resistance to four antibiotics namely;

AML, AMP, TE and C. There were 37 isolates of which high resistance was

registered in AML where 22 (60%) of isolates displayed resistance followed 20

(54%) of bacterial isolates displayed resistance to AMP. The rest was 13 (35%)

displayed resistance to Te and 10 (27%) displayed resistance to C (Figure 4.5).

Figure 4.5: Number of bacterial isolates from Masinga dam resistant to antibiotics.

The bacterial isolates from Masinga dam registered resistance to at least one

antibiotic E. coli registered resistance to AML, AMP and TE while Pleisiomonas

shigelloides registered resistance to AML and AMP. Like the isolates from the

Sagana, the antibacterial response of isolates from dam was more resistant to AML

and AMP compared to the rest of antibiotics. However, for bacterial isolates from

Masinga dam, there was no significance difference for antibacterial response from

dam (F=1.84, P=0.14). (Table 4.7).

37
Table 4.7 Antibacterial response of isolates from Masinga dam
AML CTX CIP S AMP GEN TE C NA CXM
Isolates No I
R S I R S I R S I R S I R S I R S I R S I R S I R S I R S

Citrobacter 6 5 1 0 0 6 0 0 6 0 0 3 3 5 1 0 0 6 0 1 3 2 3 3 0 0 6 0 0 6 0
freundii
Aeromonas 2 1 0 0 0 2 0 0 2 0 0 2 0 1 1 0 0 2 0 0 2 0 0 2 0 0 2 0 0 2 0
sobia
Vibrio 1 1 0 0 0 1 0 0 1 0 0 1 0 1 0 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0
vulnificus
Edwardsiella 2 0 1 1 0 2 0 0 2 0 0 2 0 0 2 0 0 2 0 2 0 0 0 2 0 0 2 0 0 2 0
tarda
Ecoli 8 2 6 0 0 6 2 0 5 3 0 6 2 2 3 3 0 6 2 2 4 2 0 4 4 0 4 4 0 8 0
Enterobacter 6 3 3 0 0 6 0 0 3 3 0 6 0 2 4 0 0 3 3 2 3 1 1 4 1 0 4 2 0 4 2
sakazakazii
Enterobacter 3 1 2 0 0 3 0 0 3 0 0 3 0 2 1 0 0 3 0 1 2 0 1 2 0 0 3 0 0 3 0
cloace
Pseudomonas 2 2 0 0 0 2 1 0 2 0 0 2 0 3 0 1 0 6 1 3 0 4 4 1 2 0 3 4 0 4 0
fluorescenes
Proteus 2 2 2 0 0 4 0 0 4 0 0 4 4 0 0 0 0 4 0 2 2 1 1 3 0 0 4 0 0 4 0
mirabilis
Enterobacter 2 2 0 0 0 1 1 0 2 0 0 1 1 2 0 0 0 1 1 0 2 0 0 1 1 0 2 0 0 1 1
aggromerans
Plesiomonas 2 2 1 0 0 2 0 0 1 1 0 1 1 2 0 0 0 1 1 0 2 0 0 1 1 0 2 0 0 2 0
shigelloides
Pseudomonas 1 1 0 0 0 0 1 0 0 1 0 1 0 0 1 0 0 0 1 0 1 0 0 1 0 0 1 0 0 1 0
aureginosa
Total 37 22 16 1 0 35 5 0 31 8 0 32 11 20 13 4 0 35 9 13 22 10 10 25 9 0 34 10 0 38 3
% 100 59 43 3 0 95 14 0 84 22 0 86 30 54 35 11 0 95 24 35 59 27 27 68 24 0 92 27 0 103 8

38
 CHAPTER FIVE

DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS

5.1 Discussion
Members of Enterobacteriacea are part of the gut flora found in the intestines of

humans and other animals, while others are found in water or soil, or are parasites on a

variety of different animals and plants. Fish is no exception and in this study most of

the baceria isolates were from the family Enterobacteriacea ; Citrobacter freundii,

Edwardsiella tarda, Enterobacter sakazakii, Enterobacter cloacae, Enterobacter

amnigenus, Enterobacter agglomerans, Escherichia coli, Klebsiella pneumoniae,

Klebsiella ornithinolytica, Proteus mirabilis, Providencia stuartii, Shigella boydii,

Salmonella spp, and Plesiomonas shigelloides. This is in line with the findings of

Ogbondeminu and Olayemi (1993) who reported that 50% of the microorganisms

recovered from both fish and water of earthen pond fertilized with animal faecal waste

were members of the family Enterobacteriaceae.

The other bacteria isolated include; Vibrio mechnikovii, Aeromonas sobia,

Pseudomonas aeruginosa, Pseudomonas fluorescenes, Chromobacterium violaceum

and Acinetobacter spp. They are widely distributed in aquatic environment and in the

soil, and have been implicated as opportunistic pathogens causing diseases in human

beings. These bacteria have been isolated in other studies by Nganou et al. (2011) who

isolated Aeromonas spp ,Vibrio spp, Pleisomnas spp, Acinetobacter spp,

Enterobacteriacea, Pseudomonas spp, from tilapia fish collected from four lakes in

Cameroon. In other studies (Naim, 2012) recovered A. hydrophila, Vibrio spp., S.

putrefaciens, Staphylococcus sp., Streptococcus spp., and Edwardsiella spp.

Aeromonas spp, Chromobacterium violaceum, Citrobacter freundii, Escherichia coli,

39
and Plesiomonas shigelloides were isolated in the gastrointestinal regions of semi-

intensively cultured tilapia, Oreochromis niloticus. Although the presence of these

bacteria is not often associated with fish diseases or enteric diseases in man, the health

implications of the introduction of these organisms into natural water via the fish

faeces in the aquaculture wastewaters should not be ignored (Naim, 2012).

There were more bacterial species isolated from the ponds than the species isolated

from the dams during the two seasons. There was correlation between the sites in

which the bacteria were isolated. This can be attributed to the fact that the ponds are

fertilized using animal manure to enhance alga bloom and also due to accumulation of

feaces of fish and left over feed in the earthen pond (Davis and Goulder, 1993;

Makosora and Jazek, 1994). During the wet seasons, lower temperatures inhibit micro

bacterial activity, the reason adduced for higher number of bacteria isolates during dry

season compared to wet season according to Wemedo (2002). Another reason given

attributed to this phenomenon is that saturation of the soil by rain, limits activity by

reducing aeration (Marshall and Devinny, 1998).

The results revealed that of all the specimens sampled sediment had the lowest number

of bacteria isolates; this is in line with an earlier study by Niemi and Taipalinen (1982)

who reported a very low count in sediment. This could be attributed to lack of sunlight

as it plays an important role in the bacteria growth (Ferguson et al., 1996). In this

study there were higher numbers of bacteria in all specimens during the dry season this

could be attributed to the fact that bacteria multiply more in high temperatures.

(Chowdhury et al., 1989) observed similar results in the intestinal bacterial load of

tilapia. Research by (Sugita et al., 1985; Markosova and Jezek, 1994) reported that

40
populations of indicator bacteria increased with increasing water temperature as

temperature becomes favorable for growth of bacteria during summer months.

The findings revealed different members of Enterobacteriacea were isolated from

both the dams and the ponds some of which are pathogenic and others are non

pathogenic. In other studies there are reports of isolation of different members of

Enterobacteriaceae, as potential fish and human pathogens from natural manured

carps, striped bass, tilapia, eel and its earthen culture environment (Nair and Nair,

1988; Karunasagar et al., 1992; Nedoluha and Westhoff, 1997; Muratori et al., 2000).

The occurrence of these pathogenic Enterobacteriaceae was more in the Sagana ponds

than the dams which could be due to use of animal manure in the ponds

(Ogbondeminu, 1993). In the present study Salmonella members of

Enterobacteriaceae, and Shigella were recovered from fish, water and water sediments

from ponds at Sagana fish ponds where they use integrated fish culture systems. This

is an indication that there exists an inherent risk of contamination by pathogens

present in environment as observed in natural manured earthen ponds by Nedoluha

and Westhoff (1997). The presence of Salmonella spp. indicates feacal contamination

of water from which the fishes were harvested.

Aeromonas sobria was one of the bacteria isolated from both sources during the two

seasons. It is a known human pathogen (Mateos et al., 1993; Thune et al. 1993.,

Austins and Adams, 1996) and therefore poses a risk of fish-borne Aeromonas

gastroenteritis in consumers of improperly cooked fish. The finding of vibrio spp

during the dry season is in line with studies carried out by (Al-Harbi and Uddin, 2003)

who found that there were more counts during the summer than during winter.

41
Pseudomonas aeruginosa was isolated during the two seasons and it is a potential

human pathogen. It can persist even after processing posing a health hazard to

consumers. Lyhs et al. (1998) reported that pseudomonas was responsible for 15.3%

of spoilage of preserved fish products, making the organism important in food

spoilage with economic losses. It is therefore that the fish from both Sagana and

Masinga, be processed and stored well to eliminate contamination.

The E .coli has been traditionally recognized as an indicator organism of faecal

contamination of water and seafood (Geldreich, 1997). Escherichia coli are normal

inhabitants of the intestinal tracts of all warm blooded animals. In this study E. coli

was recovered in all fish samples and in water samples indicating poor hygiene and

sanitary condition in Sagana and Masinga dams. Like many of landing beaches in

Kenya Masinga dam lacks proper sanitation facilities at the landing sites and this

could explain the presence of E. coli in all specimens. In other studies Chandraval et

al. (2010) found that fish and water samples collected from Nadia District of West

Bengal in India were contaminated with faecal coliforms like E. coli.

Edwardsiella tarda which was isolated from fish samples in both seasons are

considered a serious problem in tropical or subtropical areas. Infections associated

with this species include gastroenteritis, wound infections, and systemic diseases such

as septicemia, meningitis, cholecystitis, and osteomyelitis (Janda and Abbott, 1993).

Edwardsiella tarda has been isolated in fishes from fresh water aquaculture

environment and retail markets in India (Pankajkumar, 2009).

42
Citrobacter freundii and Proteus mirabilis were isolated in the Masinga dam samples

and have also been isolated in other studies (Niemi and Taipalinen, 1982; Apun et al.,

1999).

Chromobacterium violaceum is a Gram-negative rod isolated from soil and water in

tropical and subtropical regions. In this study it was isolated in Sagana ponds only.

Infections caused by C. violaceum are rare among mammals, but Apun et al. (1999)

reported two cases of human infection caused by both pigmented and non pigmented

strains of C. violaceum. Pleisiomonas shigelloides is a common pathogen in tropical

regions associated with diarrhea and occasional opportunistic infections in humans. In

this study it was isolated from Masinga dam during the dry season.

The bacterial isolates were highly sensitive to ciprofloxacin (100%), which is

gentamycin (98.9%) and this is in line with the findings of (Jawahar, 2011) whose

findings were similar with bacterial human pathogens highly sensitive to ciprofloxacin

(91%), gentamycin (85%) and chloramphenicol (88%). The relatively high resistance

to ampicillin of 61.5 % to most of the isolates is in partial agreement with the findings

by (Barat et al., 2002) who found a prevalence of 93.4% resistance of gram negative

bacteria isolated from fish to ampicillin, also the findings of Newaj-fyzul et al. (2006)

of predominance resistance to ampicillin of 90.2%. This could be due to the fact that

the use of antibiotics in aquaculture in Kenya is very limited. The finding of 31.8 %

isolates resistance to tetracycline is comparable with 47% reported by Castro-

Escarpulli et al. (2003) for isolates recovered from Tilapia (Oreochromis niloticus

niloticus) intended for human consumption in Mexico.

43
Cow dung manure serves a potential carrier of pathogenic bacteria which are capable

of transmitting zoonotic diseases to humans as a result of contact with the manure,

when this untreated manure is used to fertilize fish ponds, it may lead to increase in

bacterial infections in the fish and serves as a potential source of food borne infections

for the fish consumers. However resistance to the antimicrobial agents may be due to

indiscriminate, widespread and lengthy use of tetracycline, chloramphenicol and

gentamicin in treatment of cow infections (Omojowo and Omojasola, 2013). Sagana

fish ponds are fertilized using cow dung manure and this can explain why in the study

bacterial isolates from the ponds showed resistance to more antibiotics than those from

the dams. In another study, Andreas et al. (2002) concluded that, integrated fish

farming seems to favor antimicrobial-resistant bacteria in the pond environment. In

another study by Anja et al. (2000) found that high levels of individual and multiple

antimicrobial resistances were demonstrated within the collected Flavobacteria and

Aeromonads, thus indicating a substantial impact of fish farming on several groups of

bacteria associated with aquaculture environments.

5.2 Conclusions
a) Fish from both Sagana ponds and Masinga dam harbors pathogenic bacteria and

bacteria which are natural inhabitants of the animal gut flora. In Sagana E. coli,

Salmonella, Shigella boydii, were isolated among others while in Masinga dam

Pleisiomonas shigelloides, E. coli were isolated among others.

b) There was no significant difference of bacteria flora species isolated in the two

sites or in the two seasons. Some of the bacteria that were isolated in both seasons

were E. coli, Citrobacter freundii while Salmonella spp, Shigella boydii among

others. This shows that bacteria species found on the fish skin and in the gut are

similar to the ones found in the environments the fish is cultured in.

44
 c) The study showed that there was antibiotic resistance to the isolates with a

relatively high resistance to ampicillin of 61.5% and sensitive to ciprofloxacin

(100%), gentamycin (98.9%).

d) There was variation in antibacterial response of isolates from Sagana but no

significance difference in Masinga dam

5.3 Recommendations
Sanitary conditions under which fish are reared in ponds should be improved,

following standard or good practices; such as use of good quality water, use of feeds

with high microbial quality, regular draining of pond water after specific period of

time, closure of ponds to the public among other things. The public should be

enlightened on the inherent danger that may accompany handling of fresh fish or

consumption of improperly cooked fish. Therefore, fish must be properly cooked

before it is consumed to avoid contact with the microbes that may be associated with

it.

The use of antibiotic in fish farming and animal husbandry should be monitored. In

addition, the implication of this high level of antibiotic resistance on the choice of

antibiotics in relation to zoonotic infections should be noted and efforts should be

made to stop indiscriminate use of antibiotics.

45
Further research on farmed fish species taking into considerations different ponds and

dams from different climatology regions to determine relationship between the

microbial activities in fish and the different climatic conditions of the regions.

Moreover, the use of antibiotics in aquaculture for promotion of growth in different

climatic conditions should be studied further.

46
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 APPENDICES

Appendix 1: Overall data on specimen, isolates and the response to antibiotics

SPECIMEN NO. ISOLATE AML CTX CIP S AMP CN TE C NA CXM

ATCC 25922 E.coli S S S S S S S S S S
TS1 Aeromonas spp. R S S I R S S S S S
TG3 Citrobacter freundii R S S S R S R I S S
TS2 Kleb.ornithnilotyca R S S S R S R I S S
TG1 E.coli S S S S S S R R S S
TS3 Kleb.pneumo S S S S S S S S S S
TG1 Edwardsiella tarda R S S I R S S S S S
TG1 Kleb.pneumo R S S R R S R R S S
ST1 Kleb.pneumo R S S S R S R S S S
CS3 Kleb.pneumo S S S S S S I S S S
CS2 Aeromonas spp. R S S S R S S S S S
CS2 Citrobacter freundii R S S I R S S S S S
CS1 Providencia spp. R S S S R S S S S S
CG1 Citrobacter spp. R S S I R S S S S S
CS1 Pseudomonas spp. R S S I R S R R R R
SCG Aeromonas spp. S S S S S S S S S S
SCG Aeromonas spp. S S S S S S S S S S
MCS E.coli S S S S S S R R I S
MTS Citrobacter freundii R S S I R S R I S S
MTG Citrobacter spp. R S S S R S I I S S
MCG Edwardsiella tarda S S S S S S R I S S
MTG Citrobacter freundii R S S S R S R R I S
STG Edwardsiella spp. S S S S S S S S S S

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STG Aeromonas spp. S S S S S S S S S S
TG1 Pseudo.aeruginosa R S S I R I R R R R
STS Salmonella spp. R S S S R S S S S S
TG1 E.coli S S S S S S I I I S
TG2 Citrobacter spp. R S S I R S S S S S
CG1 Providencia spp. R S S S R S S S S S
SCG Aeromonas spp. R S S S R S S S S S
MCS Ent.agglomerans R S S S R S S I S S
STS Citrobacter spp. R S S I R S I I S S
STG Edwardsiella tarda S S S S S S S S S S
SCS Proteus spp. R S S S R S R R S S
STS Aeromonas spp. R S S S R S S S S S
CS3 Citrobacter spp. R S S I R S S S S S
STG Proteus mirabilis R S S S R S S S S S
STG Providencia stuartii S S S S S S R I S S
CS1 Enterobacter spp. R I S S R S I I S S
STS Vibrio metchnikovii R S S I R S S S S S
MTS Proteus spp. R S S S R S I I S R
MCS Citrobacter freundii R S S S R S I R S S
STG Chromo. Viobceum S I S S S S R R I I
MCS Aeromonas spp. R S S S R S S S S S
CS3 Enterobacter spp. R S S S R S S S S S
STS Pseudomonas spp. R I S R R S R R R R
SCG Providencia spp. R S S S R S R R S S
SCS Citrobacter spp. R S S S R S R R S S
MCS Ent.sakazakazii S S S S S S S S S S
MTG Proteus mirabilis R S S S I S I R S S
SCG Shigella boydii R S S S R S I S S S
SCS Pseudo.aeruginosa R S S S R S I I S S

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SCS Pseudomonas spp. R S S S I S I I S S
MTG Ent.sakazakazii R S S S R S I R I S
MTG Ent.amnigenus R S S S R S S S S S
SCG Citrobacter freundii S S S S S S S S S S
SCS Edwardsiella tarda S S S S S S S S S S
MCS Enterobacter spp. R S S S R S S S S S
ATCC 25922 E.coli S S S S S S S S S S
MCG Citrobacter freundii S S S S S S R R I S
MTS Ente. cloacae R S S S R S R R S S
MTG Vibrio vulfinificus R S S S R S S R S S
Dam water Pseudo fluoresc/putida R S S S R S R R S S
STS Aeromonas sobria R S S S R S S S S S
MCG E.coli I S S S I S R I S S
Sagana water Acinetobacter spp S S S S S S I S S S
MTG Enterobacter spp. R S S I R S R R I S
MCG Edwardsiella spp. I S S S I S R R S S
Sagana water Acinetobacter spp S S S S S S R R S S
Sagn water E. fergusonii R S S I R S S S S S
STS Pseudo.aeruginosa R S S S R S I R R S
STG Citrobacter freundii R S S S S S I R S S
STG Aeromonas sobria R S S I R S S S S S
STG Salmonella spp.(STM) S S S S S S S S S S
MCS Enterobacter spp. R I S S R S I I S S
Sagana water Citrobacter spp. R S S S R S I I S S
MTG Ent.amnigenus R S S S R S R R S S
MCS Plesio.shigelloids S S S S S S S S S S
STG Pseudo.aeruginosa R S S S S S I R S S

61