Microbial Quality and Antibiotic Residues of

Fish Sold in the Gaza strip, Palestine.

الجودة الميكروبية ومتبقيات المضادات الحيوية لألسماك المباعة في قطاع غزة، فلسطين.

By

Asmaa El Siqali

Supervised by

Prof. Dr. Abdelraouf A. Elmanama Dr. Kamal J. Elnabris

A thesis submitted in partial fulfilment

of the requirements for the degree of

Master of Biological Sciences- Microbiology

October 2017

The Islamic University of Gaza

Deanship of Research and Postgraduate

Faculty of Science

Master of Biological Sciences

بغــزة اإلســـــالميــةـة ـــــــــــامعـالج

لعلمي والدراسات العلياالبحث ا عمادة

ة العلــــــــــــــــــــــــــــــــــــوم ليــــــك

اجســــــتير علــــــــــــــــــــوم حياتيةم

I

Declaration

I, the undersigned hereby, declare that the thesis titled:

Microbial Quality and Antibiotic Residues of Fish Sold in the

Gaza strip, Palestine.

Declaration

I understand the nature of plagiarism, and I am aware of the University’s policy on

this.

The work provided in this thesis, unless otherwise referenced, is the researcher's own

work, and has not been submitted by others elsewhere for any other degree or

qualification.

Asmaa El Siqali Student's name:

Asmaa El Siqali Signature:

2017/11/8 Date:

I

Abstract

Introduction: Fish diseases caused by pathogens (e.g. bacteria, viruses, fungi and

parasites) affect the survival and growth rates of fish, and consequently lead to major economic

losses. Furthermore, the microorganisms responsible for these infections belong to bacterial

families that also produce infections in humans. Therefore, their transmission to human is

highly probable. Several antibiotics including; oxytetracycline, sulfamerazine and ormetoprim,

are used for treating bacterial infections in farmed fish. The use of antibiotics in aquaculture

systems is usually associated with serious health hazard not encountered in wild captured

species. The main concern is antibiotic residues and development of antimicrobial resistance in

bacteria that may be transferred to consumers. Several types of fish are consumed daily by

inhabitants of Gaza strip as source of protein.

Objectives: In this study, the microbial quality for locally farmed, caught and imported (frozen)

fish was evaluated and the presence of antibiotic residues was investigated.

Methodology: The study examined 100 fish specimens that were purchased from local markets

(60 farmed and 30 frozen and 10 caught fish). Total coliform, total viable count, Staphylococcus

aureus, Salmonella, and Vibrio spp. were tested using standard methods. To investigate the

presence of antibiotic residues, four classes of antibiotics were determined in fish samples using

a bioassay method recommended by United States Department of Agriculture (USDA).

Results: The most detected antibiotic residues were aminoglycosides 52 (52%) in sea bream,

red tilapia and Nile tilapia. followed by tetracyclines 1 (1%) in sutchi catfish fillet and negative

results for β-lactams and macrolides. Microbiological quality tests showed that 39% of fish

samples failed to comply with the Palestinian standards, the percentage of failure due to Total

Plate Count (4%), Total Coliform bacteria (39%), S. aureus 13%, and Salmonella spp. (1%).

Conclusions: Results confirmed the presence of antibiotic residues in fish samples collected

from Gaza strip. A confirmatory method such as gas chromatography (GC) is recommended to

be used to determine residues compliance with the maximum residue limits. It is also

recommended that measures should be implemented to ensure observing proper withdrawal

periods before marketing and drug control in veterinary use. In addition, a monitoring policy

should be implemented to ensure the conformity of fish sold in Gaza strip with international

standards. The results emphasizes the need to promote awareness about possible health hazards

that could result from poor handling of farmed fish.

Key words: Fish, Microbial quality, Antibiotic residues, Gaza-Palestine.

II

ملخص الدراسة

من أكبر مثل البكتيريا والفيروسات والفطريات والطفيلياتاملختلفة املمرضاتأمراض األسماك التي تسببها تعتبر مقدمة:

نات الحية الكائ وفي هذا السياق تعتبرعلى معدالت بقاء ونمو األسماك، مما يؤدي إلى خسائر اقتصادية كبيرة. املؤثرات

مما يزيد يضا،أ البشر بيناألسر البكتيرية التي تنتج العدوى ذات إلى تنتميبين األسماك، و دوى عالالدقيقة مسؤولة عن

العديد من خدام يتم است البكتيرية في األسماك املستزرعةوملعالجة العدوى كبير. بشكلانتقالها إلى اإلنسان من احتمالية

ية وأورميتوبريم. وعادة ما يرتبط استخدام املضادات الحيو ،أوكسيتيترايكلين، سلفامرازين ، وتتضمناملضادات الحيوية

البيئات الطبيعية لنمو األسماك، إال أن مصدر القلق في ال تحدث عادة بمخاطر صحية كبيرةفي نظم االستزراع املائي

قاومة تلك ماك ملريا في أجسام األسيوالعناصر املناعية التي طورتها البكتاملضادات الحيوية تلك بقايا االساس هنا هو

لبروتين.ل مهم عدة أنواع من األسماك يوميا كمصدرقطاع غزة ستهلك يللمستهلكين. و انتقالهاالتي يمكن و املضادات

وردة املست واألسماكمحليا طادةواملصلألسماك املستزرعة يةجودة امليكروبال: تم في هذه الدراسة تقييم األهداف

.في تلك األسماك ايا املضادات الحيويةجود بقباإلضافة إلى و )املجمدة(

مجمدة 01مستزرعة ومنها 01عينة من األسماك تم شراؤها من األسواق املحلية ) 011 قامت الدراسة بفحص: املنهجية

، كتيرياالعدد الكلي للب، البكتيريا القولونيةإجمالي :باستخدام الطرق القياسية التالي تم اختباروقد ،(مصطادة 01و

ن وجود بقايا املضادات الحيوية، تم تحديد أربع فئات م . ولقياس مدىالفيبروكورات العنقودية الذهبية، الساملونيال، و امل

وزارة الزراعة األمريكية حسب توصيات املقايسة الحيويةاملضادات الحيوية في عينات األسماك باستخدام طريقة

)أوسدا(.

سمك الدنيس( في %25) 25أمينوغليكوزيدات التي تم رصدها هيضادات الحيوية امل أنواع متبقياتكانت أكثر النتائج:

-ابيتالنتائج السلبية كما كانت ،( في فيليه سمك السلور %0) 0التتراسيكلين ويلي ذلك ،والبلطي األحمر والبلطي النيلي

نات األسماك لم تمتثل للمعايير من عي %03كتامز واملاكروليدات. وأظهرت اختبارات الجودة امليكروبيولوجية أن ال

لمكورات العنقودية ل %00ريا الكلية القولونية، ويلبكتل %03، وريا الكليةيلحساب البكت %4وذلك بنسبة الفلسطينية،

لساملونيال.ل %0 و ،الذهبية

ي لذا توص ،ةأكدت النتائج وجود بقايا املضادات الحيوية في عينات األسماك التي تم جمعها من قطاع غز :الخالصة

الدراسة املضادات الحيوية تلك بقايا التزام مستويات( لتحديد مدى GC) الجازكروماتوجرافيدة مثل باستخدام طريقة مؤك

بة لضمان مراعاة فترات االنسحاب املناسالالزمة تدابير الأيضا بتنفيذ الدراسة وص ى ت. و املسموح بها القصوى بالحدود

ة األسماك لضمان مطابق رقابةينبغي إضافة إلى ذلك تنفيذ سياسة و . ةالبيطري األدوية اماستخد قبل التسويق ومراقبة

على ضرورة تعزيز الوعي بشأن املخاطر الصحية املحتملة الدراسة نتائج وتؤكداملباعة في قطاع غزة مع املعايير الدولية.

التي يمكن أن تنجم عن سوء معالجة األسماك املستزرعة.

.فلسطين ،غزة ،املضادات الحیویة متبقيات، بيةجودة املیکرو الاألسماك، :فتاحيةامل الكلمات

III

DEDICATION

To my great mother

To pure soul of my father

To my whole family

To my best friends

I dedicate this work.

IV

ACKNOWLEDGEMENTS

I would like to express my gratitude to my supervisors Prof. Dr. Abdelraouf A.

Elmanama and Dr. Kamal J. Elnabris for the useful comments, remarks and

engagement through the learning process of this master thesis.

Furthermore, I would like to thank Mr. Mohammed Albayoumi for introducing

me to the topic as well for the support on the way.

Also, I like to thank Public health laboratory staff, who have willingly shared

their precious time during the process of searching.

I would like to thank my loved ones, who have supported me throughout entire

process, both by keeping me harmonious and helping me putting pieces together.

I will be grateful forever for your love.

V

Table of Content

Abstract ........................................................................................ I

DEDICATION ............................................................................. III

ACKNOWLEDGEMENTS ............................................................ IV

Table of Content ............................................................................ V

List of Tables ............................................................................. VIII

List of Figures .............................................................................. IX

Chapter I Introduction .................................................................... 2

1.1 Overview ............................................................................... 2

1.2 Objectives ............................................................................. 5

1.2.1 General objective ..................................................................................................... 5

1.2.2 Specific Objectives .................................................................................................. 5

1.3 Significance of the Study .......................................................... 5

Chapter II Literature Review ............................................................ 9

2.1Introduction ........................................................................... 9

2.2 Importance of fish ................................................................... 9

2.3 Local Fish production and consumption ...................................... 9

2.4 Local sources of fish .............................................................. 10

2.5 Aquaculture ......................................................................... 11

2.6 Antimicrobials...................................................................... 12

2.6.1 Definition of antimicrobials ................................................................................... 12

2.6.2 Antimicrobials use in fish culture .......................................................................... 12

2.6.3 Antibiotics administration route and fate ............................................................... 13

2.6.4 Harmful effects of antimicrobials use .................................................................... 14

1.4.6.2 Mechanisms of development of antibiotic resistance ......................................... 14

2.6.5 Effects of antibiotic on public health ..................................................................... 14

2.6.6 Adverse environmental impacts ............................................................................. 16

2.6.7 Exposing other (non-target) animals that may act as food for humans to antibiotics

......................................................................................................................................... 17

2.6.8 Adverse Ecological impacts ................................................................................... 17

2.7 Antimicrobial residues ........................................................... 18

2.7.1 Definitions .............................................................................................................. 18

2.7.2 Harmful effects of antimicrobial residues .............................................................. 18

2.7.3 Factors contribute to the drug residue problem ..................................................... 19

VI

2.7.4 Screening technique for the detection of antimicrobial residues in food products 20

2.7.5 Confirmation methods ........................................................................................... 22

2.7.6 Microbiological inhibition tests ............................................................................. 22

2.7.7 Examples of microbiological assay methods ......................................................... 23

2.7.8 Other tests .............................................................................................................. 24

2.8 Residue Control Programs ...................................................... 24

2.9 Withdrawal period ................................................................ 24

2.10 Previous studies for antibiotic residues .................................... 24

2.11 Fish Microbial quality .......................................................... 25

2.12 Microbial indicators ............................................................ 28

2.12.1 Staphylococcus aureus ......................................................................................... 28

2.12.2 Salmonella spp. .................................................................................................... 29

2.12.3 Total plate count .................................................................................................. 29

2.12.4 Coliform bacteria ................................................................................................. 30

2.12.5 Vibrio spp. ............................................................................................................ 30

2.12.6 Previous studies for microbial quality ................................................................. 30

Chapter III Materials and Methods .................................................. 34

3.1 Materials ............................................................................. 34

3.1.1 Equipment .............................................................................................................. 34

3.1.2 Microorganisms, media and reagents .................................................................... 34

3.2 Study area ........................................................................... 35

3.3 Fish collection ...................................................................... 36

3.4 Fish transport and handling .................................................... 36

3.5 Antibiotic residues examination ............................................... 37

3.5.1 Principle of the test ................................................................................................ 37

3.5.2 Buffer preparation .................................................................................................. 37

3.5.3 Sample preparation and storage ............................................................................. 38

3.5.4 Preparation of bacterial suspensions: ..................................................................... 38

3.5.5 Preparation of Bioassay Plates ............................................................................... 39

3.5.6 Assay procedures: .................................................................................................. 40

3.5.7 Results interpretation ............................................................................................. 41

3.6 Microbial analysis ................................................................. 42

3.6.1 Sample preparation ................................................................................................ 42

3.6.2 Total viable count (TVC) ....................................................................................... 42

3.6.3 Total coliform count .............................................................................................. 43

3.6.4 Staphylococcus aureus ........................................................................................... 43

VII

3.6.5 Detection of Salmonella ......................................................................................... 43

3.6.6 Detection of Vibrio spp. ......................................................................................... 44

3.7 Questionnaire ....................................................................... 45

3.8 Data analysis ........................................................................ 45

Chapter IV .................................................................................. 47

Results ........................................................................................ 47

4.1 Biometric measurements ........................................................ 47

4.2 Detection of antibiotic residues ................................................ 48

4.2.1 Aminoglycosides residues ..................................................................................... 48

4.3 Microbiological indicators ...................................................... 49

4.3.1 Total plate count .................................................................................................... 52

4.3.2 Total coliform ........................................................................................................ 54

4.3.3 Staphylococcus aureus ........................................................................................... 56

4.3.4 Salmonella .............................................................................................................. 57

4.3.5 Vibrio spp. .............................................................................................................. 57

4.4 Questionnaire results ............................................................. 57

4.4.1 Use of Antibiotics in Surveyed Farms .................................................................. 58

Chapter V Discussion .................................................................... 60

5.1 Antibiotic residues ................................................................ 60

5.2 Microbial quality .................................................................. 64

5.2.1 Total plat count ...................................................................................................... 64

5.2.2 Total coliform ........................................................................................................ 65

5.2.3 S. aureus ................................................................................................................. 65

5.2.4 Salmonella spp. ...................................................................................................... 65

5.2.5 Vibrio spp. .............................................................................................................. 66

5.3 Questionnaire analysis ........................................................... 66

Chapter VI Conclusions and recommendations .................................. 68

6.1 Conclusions ......................................................................... 68

6.2 Recommendations ................................................................. 69

References ................................................................................... 71

VIII

List of Tables

Table (2.1): Demonstrates advantages and disadvantages of different screening

methods of residues analysis (Sirdar, 2011). .......................................... 21

Table (2.2): Agar diffusion tests used for the screening of antimicrobial residues

in meat. ....................................................................................... 22

Table (3.1): Equipment used in the study ............................................. 34

Table (3.2): Microorganisms, media and reagents ................................... 35

Table (3.3): Glassware and disposables used in experimental work ............. 35

Table (3.4): Preparation of Phosphate Buffers with different pH values ........ 37

Table (3.5): Bacterial suspension concentrations in plates ......................... 39

Table (3.6): The pH and Antibiotic disks according to plate number assigned

for the Bioassay ............................................................................. 41

Table (3.7): Interpretation of results of five-plate bioassay (USDA, 2011) .... 42

Table (4.1): List of fish species collected from local markets and farms with

means, standard deviations and ranges of weight (g) and length (cm). .......... 47

Table (4.2): Antibiotics detected in different fishes with prioritization of

antimicrobials categorized as Critically Important and Highly Important. ...... 48

Table (4.3): Aminoglycoside residues screening results of analyzed Fish ...... 49

Table (4.4): Ranges of total plate count, total coliform count and S. aureus

count expressed as colony forming unit (cfu/g) in frozen, wild caught and

farmed fish species. ........................................................................ 50

Table (4.5): Number of positively and negatively contaminated fish, the

prevalence of bacterial contamination and the occurrence of multiple

contamination in the different types of fish ............................................ 51

Table (4.6): Number and percentage of negativly and positevely (single, double

and triple contamination) contaminated fish species. ................................ 52

Table (4.7): The compliance with the Palestinian microbiological standard for

fish. ............................................................................................ 52

Table (4.8): The compliance with total plate count (TPC) standards of frozen,

wild caught and farmed fish species. ................................................... 53

Table (4.9): The compliance with total coliform standards of frozen, wild

caught and farmed fish species. .......................................................... 55

Table (4.10): The compliance with S. aureus standards of frozen, wild caught

and farmed fish species. ................................................................... 56

Table (4.11): Farmer's responses to the questionnaire. ............................. 58

Table: (4.12): farmer's behaviors in dealing with antibiotics in farms .......... 58

IX

List of Figures

Figure (3.1): Lyophilized bacterial strains that was used in Inhibition assay

(Microbiologics, 2017) .................................................................... 39

Figure (3.2): Standard stainless steel bioassay cylinders ........................... 40

Figure (3.3): Stainless steel bioassay cylinders showing variation in zones of

inhibitions .................................................................................... 40

Figure (4.1): Comparison of percentages of farmed, frozen imported and wild

caught fish which were found to contain aminoglycosides residues. ............. 49

Figure (4.2): Number and percentage of negativly and positevely (single,

double and triple contamination) contaminated fish based on their type. ....... 51

Figure (4.3): Percentage of fish that complied with the Palestinian Standards for

TPC. ........................................................................................... 53

Figure (4.4): Number and percentage of the different types of fish that pass and

fail the Palestinian Standards for TPC. ................................................. 54

Figure (4.5): Percentage of fish sample that compiled the Palestinian standard

for total coliform. ........................................................................... 54

Figure (4.6): Number and percentage of the different types of fish that pass and

fail the Palestinian Standards for total coliform. ...................................... 55

Figure (4.7): Percentage of fish that complied with the Palestinian Standards for

S. aureus. ..................................................................................... 56

Figure (4.8): Number and percentage of the different types of fish that pass and

fail the Palestinian Standards for S. aureus. ........................................... 57

X

List of Abbreviations

Allowed Daily Intake ADI

Acceptable Daily Intake ADI

Antimicrobial Residues AMR

Alkaline Peptone Water APW

American Type Culture Collection ATCC

Calf antibiotic and sulfa test CAST

Colony Forming Unit CFU

Eicosapentenoic Acid EPA

European Union EU

Food-borne diseases FBD

Food and drug administration FDA

Four plate test FPT

Food Safety Inspection Services FSIS

Gas chromatography GC

Heterotrophic Plate Count HPC

High Performance Liquid Chromatography HPLC

International Commission on Microbiological Specifications Food ICMSF

Liquid Chromatography LC

Maximum Residue Limit MRL

Maximum Residue Limits MRLs

Oxytetracycline OTC

Rappaport-Vassiliadis broth RV

Staphylococcal Enterotoxins SEs

Total Coliform TC

Thiosulphate Citrate Bile Salt Sucrose TCBS

Thin Layer Chromatography TLC

Total Plate Count TPC

Tetrathionate Broth TT

Total Viable Count TVC

Violet Red Bile Agar VRBA

World Health Organization WHO

Xylose lysine desoxycholate

XLD

2

Chapter I

Introduction

1

Chapter I

Introduction 1.1 Overview

Fish is an essential source of food for people and is considered as very good source of animal

protein consumed by the world’s population (Houlihan, Boujard, & Jobling, 2008). It

constitutes an important component of the overall diet of Palestinian people. The daily average

consumption of fish by people in the Gaza strip for example was found to be about 12.0g per

person, and total local consumption is about 6500 tons per annum (Elnabris, Muzyed, & El-

Ashgar, 2013).

The high nutritional quality of fish is also attributed to its content of essential nutrients such as

essential minerals (sodium, potassium, calcium, magnesium, phosphorus, sulphur, iron,

manganese, zinc, copper, and iodine) (Mogobe, Mosepele, & Masamba, 2015), and fatty acids

(eicosapentenoic acid and docosahexenoic acid) that are significant for healthy growth and

normal maintenance of the human body (Hossain, 2011).

People who eats fish as part of an overall healthy diet generally have a reduced cholesterol

levels, less incidence of heart disease, stroke and premature delivery, and more protected from

mineral deficiency diseases. Due to its ever-increasing consumption demand, fish and fishery

products also constitute an important item of trade and provide a source of income for large

number of people (Harris, 2004). Therefore, it is important to maintain the quality of fish to

attract customers and to avoid the health risks associated with poor quality fish.

The sources of fish consumed by people in the Gaza strip include wild caught fish from the

Mediterranean Sea, cultured fish as well as imported frozen and salted fish from Occupied

Palestinian territories, West Bank and different countries all over the world (Minstry of

agriculture, 2016). Imported, mainly the frozen fish represents the major source of fish in Gaza

strip. In 2015, about 60% of the total supply of edible fishery products in the Gaza strip was

from imports (Ministry of agriculture, 2016). Majority of the frozen fishes are sold in fridges,

which are mostly opened, thus exposing fish to various microorganisms some of which

compromise the shelf life of the product and/or safety in humans.

Over the past years, in order to meet the growing demand of fresh fish in Gaza strip, there have

been increased interest in the aquaculture sector in terms of productions and species

3

diversification. This is exactly true for the production of three species of fish e.g. Gilthead sea

bream (Sparus aurata), Nile tilapia (Oreochromis niloticis), and Hybrid red tilapia

(Oreochromis mossambicus × Oreochromis niloticus) (Minstry of agriculture, 2016).

In their natural habitat, fish are exposed to a wide variety of bacteria, most of which are able to

cause illness. Fish may also harbor pathogenic bacteria, which form part of their habitat. Such

pathogenic bacteria are introduced to fish because of habitat contamination (mainly fecal

contamination of the marine environment). The main source of pollution in the coastal zone of

Gaza is the discharge of untreated wastewater along the shoreline. Pollution of the coastal zone

and seawater, deteriorate the natural resources and natural habitats and diminish fish

populations. There are also indications that the quality of fish is influenced by coastal pollution

(Ministry of Environmental affairs (MEnA, 2001)).

Kumar, Rao, and Haribabu (2014), classified the bacterial pathogens associated with fish into

nonindigenous bacterial pathogen and the indigenous bacterial pathogens. While the non-

indigenous contaminate the fish or the habitat one-way or the other, the indigenous bacterial

pathogens are found naturally living in the fish’s habitat (Rodricks, 1991).

The microbial load of live and newly caught fish is carried on the slime layer on the surface of

the skin, in the gastrointestinal tract and in the gills. The bacteria from fish become harmful

when fish are physiologically disturbed, nutritionally deficient, or in the presence of other

stressors, i.e., poor water quality, overstocking, which help opportunistic bacterial infections to

appear (Austin, 2011).

Human bacterial pathogens associated with fish include Mycobacterium, Streptococcus spp.,

Vibrio spp., Aeromonas spp., Salmonella spp. and others (Lipp & Rose, 1997) (Novotny,

Dvorska, Lorencova, Beran, & Pavlik, 2004). Pathogens from fish can enter seafood chain due

to low standards of hygiene and sanitation during fish processing and during wrong treating or

storage. These harmful microorganisms may be transferred to people by ingestion of

inadequately cooked food or the handling of the fish, may pose serious health risks to human,

and might lead to food borne illnesses like, dysentery, diarrhea, typhoid, fever, salmonellosis

and cholera (Jacob & WHO Organization, 1989).

6

Detection of microbial quality in fish products is an essential to recognize and prevent problems

related to health and safety. Accordingly, the present study investigated the presence and the

levels of microbiological indicators of the edible parts of fish products presented for direct

human consumption in the local markets of the Gaza strip. This is in order to highlight the

hygienic quality of the fish sold in Gaza strip and to predict the hazard for consumers’ health

from the presence of these microorganisms.

Fish, especially farmed fish may be exposed, legally or illegally to a wide range of chemicals.

Because of the high stocking densities, especially, in intensive aquaculture systems (the system

commonly used in Gaza strip), fish are under constant attacks by a vast array of pathogens,

which increases the demand for chemical treatment, especially by antibiotics. In addition to

their therapeutics action in the veterinary field, antibiotics are also have prophylactics and

growth promoting actions. The extensive administration of antibiotics to fish, destined for

human consumption however, has become a serious problem because their residues can persist

in edible animal tissues (Romero, Feijoó, & Navarrete, 2012).

The occurrence of these residues may be due to either failure to observe the withdrawal periods

of each drug, extra-label dosages for animals, contamination of animal feed with the excreta of

treated animals, or the use of unlicensed antibiotics (Paige, 1994). Antibiotic residues in foods

of animal origin may be the reason behind many health concerns in humans. Some antibiotics

may be directly toxic or be a source of human pathogens that are resistant to different types of

antibiotics. This resistance may represent a potential danger to human health, and can cause

more dangerous sickness, prolonged hospital care and increased medical costs. Other troubles

caused by antibiotic residues include, immunopathological effects, carcinogenicity (e.g.,

sulphamethazine, oxytetracycline, and furazolidone), mutagenicity, nephropathy (e.g.,

gentamicin), hepatotoxicity, reproductive disorders, bone marrow toxicity (e.g.,

chloramphenicol), and allergy (e.g., penicillin) (Nisha, 2008).

Palestine neither has regulation, nor has veterinary supervision and control regarding the

microbial quality and antibiotic usage in animal-production for food. The misuse of veterinary

drugs as well as violative residues of antimicrobials in food of animal origin have been reported

previously in Gaza strip (Elmanama & Albayoumi, 2016).

5

To protect human health however, the safe maximum residue limits (MRLs) for drugs and other

veterinary substances (for the use as veterinary medicinal products in foodstuffs of animal

origin entering the human food chain) have been estimated by the European Union (EU Council

Regulation 2377/90/EC, 1990), and other regulatory agencies around the world, such as the

U.S. Food and drug administration (FDA) (D'Mello, 2003).

Currently, there is no data regarding the microbial quality and antimicrobial residues of locally

consumed fish products in Gaza strip. Therefore, the present study was carried out to investigate

the microbial quality and to determine the presence of antibiotic residues in the edible parts of

some fish products available for human consumption in local markets in Gaza strip.

1.2 Objectives

1.2.1 General objective

To investigate the microbial quality and the presence of antibiotic residues in the edible parts

of some fish products presented for direct human consumption in the local markets of the Gaza

strip to determine whether fish products consumed in Gaza strip are suitable for human

consumption

1.2.2 Specific Objectives

The specific objectives of this study are:

1. To address the notable deficiency of data related to bacteria associated with fish

available for human consumption in Gaza strip.

2. To determine whether the microbial load in examined fish were within the

acceptable limits

3. To determine whether there are detectable antibiotic residues in fish of Gaza strip.

1.3 Significance of the Study

Fish are important sources of both food and income to many people around the world. The

microbial contamination and antibiotic residues in fish consumed by human beings have

implications on health of human beings. For safe consumption and successful marketing,

studies that are directed toward evaluation of safety and quality standards of fish are necessary.

There are no previous studies could be found on microbial quality and antibiotic residues in

fish in Gaza strip. Therefore, the findings of this research work will provide valuable, baseline

4

information on the occurrence of microbial contamination and antibiotic residues in various

kinds of fish consumed by people in Gaza strip.

The study also will provide vital information for fish farmers, fishermen, fish handlers, vendors,

consumers, health workers, academic institutions, researchers, students, local government units,

policy makers and other stakeholders in the fish industry.

1. The stakeholders will be enlightened on the levels of microbial contamination and antibiotic

residues in fish products and their effects on human.

2. The stakeholders will be also enlightened on the necessity to ensure that they carry out

proper management of their fish products to avoid pathogens that could affect both the fish

and humans.

3. Having knowledge about the extent of microbial contamination in certain fish will

contribute greatly in formulating policies on fish products to fishermen, fish farmers, sellers

as well as consumers.

4. The outcome of the study will be beneficial to consumers, as they will become aware of the

quality of the fish they consume, thus enhancing their knowledge and skills on food safety.

5. The information may also be used in potential educational programs to educate fish farmers

on antibiotics and their effects on fish and human to prevent associated health risk impacts

to consumers.

6. The result will also educate fish farmers on the best method to use antibiotics to reduce fish

exposure to violative level of antimicrobial residues.

7. Being a public health concern, the information is vital to the general population to ascertain

the safety of the fish consumed by Palestinian people.

8. Data from this study could be used by hygiene officers and food handlers in improving and

strengthening hygienic production of fish products.

9. The result of the study will make hygiene officers more vigilant in their duty thereby helping

to decrease foodborne illness among public and related consequences by increasing

awareness of consumers and producers.

10. The result of the study will be the basis of local authority officials to intensify the

implementation of policies for issuing permits to all fish-related businesses.

11. The outcome of the research will be also beneficial to fish vendors, handlers and fishermen

for they will be directed toward proper handling and preparation of foods thereby making

them an active participant in disease prevention.

12. The information in this area can be used by researchers and students as a foundation for

further researches associated with the study.

7

13. The result of the study will also serve The Islamic University of Gaza (IUG) in achieving

their goals in strengthening the university's role in serving and developing the society

through addressing issues and problems of public concerns.

14. Finally, the findings will form the basis of recommendations which if implemented will

improve sanitation programs.

8

Chapter II Literature Review

9

Chapter II

Literature Review

2.1 Introduction

Fish and seafood products an essential food source for large numbers of world population. They

occupies the second rank after meat and poultry as staple animal protein foods where fish forms

a cheap source of protein (Houlihan et al., 2008). Fish may serve as a transporter for the vast

majority of known pathogenic bacteria, like Salmonella and Vibrio spp. (Huss, 1997). A review

of related literature, description of methods used, results, and a discussion of the results follow.

2.2 Importance of fish

The developing human populace has expanded the requirement for sustenance supply since they

are great protein sources, the interest for fish and shellfish items has expanded. Around the

world, individuals acquire around 25% of their animal protein from fish and shellfish

(Bahnasawy, Khidr, & Dheina, 2009). In 2004, around 75% (105.6 million tons) of assessed

world fish generation was utilized for coordinate human utilization (Fisheries, 2006). It has

been anticipated that fish utilization in developing nations will increase by 57 percent, from

62.7 million tons in 1997 to 98.6 million in 2020 (Retnam & Zakaria, 2010). The significant of

fish in human food is not just in its content of high-quality protein, but also to the two kinds of

omega-3 polyunsaturated fatty acids: eicosapentenoic acid (EPA) and docosahexenoic acid

(DHA). Omega-3 (n-3) fatty acids are very important for normal growth where they reduce

cholesterol levels and the incidence of heart disease, stroke, and preterm delivery (Castro-

González & Méndez-Armenta, 2008). Fish also contain vitamins and minerals which play

essential role in human health (Mogobe et al., 2015).

2.3 Local Fish production and consumption

The Palestinian fishing sector is an important economic sector. It participates in supporting the

Palestinian national economy through the employment of large numbers of fishermen. It is

estimated by the Ministry of Agriculture in November 2016 to be about 3600 fishermen and

about 500 persons engaged in fishing related activities including; fish, mechanics, electricians,

boatmen, fishing equipment traders, etc... In addition to its importance in supporting Palestinian

food security by providing animal protein from fish. During 10 years (2006-2015) interval, the

contribution of fish to the diet of Palestinian people has reached a record of about 4 kg per

person on average and ranged from 3.3 to 5.1 kg/year (Ministry of agriculture, 2016).

21

2.4 Local sources of fish

In Palestine, fish production is from both internal (which include caught and farmed fish) and

imported sources with imported fish representing the major source.

1- Farmed fish

In recent times, the popularity of fish farming has been increasing by farmers, fishermen,

investors and some hobbyists, starting with ponds, small fish farms and large farms to provide

the market with this important commodity and achieve financial benefits. The most important

farmed fish in Gaza strip are:

Gilthead sea bream (Sparus aurata): from the Sparidae family, and is one of the important

fish species cultivated in the Mediterranean region. Since 2005 there has been a considerable

increase in sea bream (S. aurata) culture in Gaza strip with the establishment of 3 rearing

facilities. The interest for fresh sea bream in Gaza strip markets has increased significantly over

the last years. The white meat, light flavor and low fat content of sea bass are major desirable

characteristics sought by the consumer.

Tilapia is a large genus belong to the Cichlidae family and comprises different species.

recently, the demand to cultured Tilapia increase as a whitefish alternative. Most Tilapia is

farmed and treated in Asia - most notably in China, Taiwan and Indonesia. Africa, Central

America and South America are other major harvesting and processing locations (Boyd, 2004).

The shape and colouration of Tilapia species vary - but in general they have a short, wide body

with long dorsal fins and spines. The most common farmed Tilapia species are the Nile Tilapia

(black) and the Mozambique Tilapia (red). Wild Tilapia can grow up to 18 pounds - but farmed

Tilapia are generally harvested when they reach between 2 - 5 pounds (Griffiths & Picker,

2011).

2- Frozen fish

Argentine hake (M. hubbsi) which sold as headless and gutted, croaker fish (M. furnieri) and

sutchi catfish fillet (P. hypothalamus also known as P. Pangasius) are originally imported from

Argentine, Uruguay and Vietnam, respectively. Where there is increasing demand to buy

because of cheap prices and good taste. In 2015, imported fish reached 5220 tons of the most

important imported frozen fish: Argentine hake (Merluccius hubbsi). Lives in Argentina.

Southwest Atlantic: off southern Brazil to Argentina and the Falkland Islands Marine (Ciarlo,

Boeri, & Giannini, 1985) it is sold as headless and gutted in Gaza markets.

Sutchi catfish fillet (Pangasius hypophthalmus) (family Pangasiidae) is a species of shark

catfish (family Pangasiidae) native to the rivers of Southeast Asia. It is mainly produced in

Bangladesh, Vietnam, Malaysia, Indonesia, Laos, Cambodia and China with Vietnam is the

22

largest Pangas-producing country (Phan et al., 2009). This fish species is also known as

Pangasianodon hypophthalmus, sutchi catfish, striped catfish or tra fish and has been widely

exported due to great acceptability, affordable cost, and white meat (Guimarães et al., 2016).

Sutchi catfish is sold as frozen fillets without skin and bone. Currently, sutchi catfish fillets

have been exported to over 80 countries worldwide including Palestine.

Croaker fish (Micropogonias furnieri) family(Sciaenidae (Croakers)) The Whitemouth

croaker lives in the western Atlantic, ranging from the Greater Antilles throughout the southern

Caribbean to the coast of South America Extending from Venezuela to Uruguay, the fishing

grounds for this economically important species include Trinidad, where it is caught mainly by

trawling (Manickchand-Heileman & Kenny, 1990).

3- Wiled caught

Mugil cephalus the flathead grey mullet (Mugil cephalus) is an important food fish species in

the mullet family (Mugilidae). It is found in coastal tropical and subtropical water worldwide.

Grey mullet is caught from Gaza Sea and soled in local markets.

2.5 Aquaculture

The broad term “aquaculture” refers to the breeding, rearing, and harvesting of animals and

plants in all types of water environments including ponds, rivers, lakes, and the

ocean. Aquaculture is established for producing seafood for human consumption; enhancing

wild fish, shellfish, and plant stocks for harvest; restoring threatened and endangered aquatic

species; rebuilding ecologically important shellfish habitat; producing nutritional and

industrial compounds; and providing fish for aquariums (Sapkota et al., 2008). In addition,

aquaculture contribute in:

1- Meeting global demands for protein

Recently, the World Fish Center has reported that the wild fish stock around the world is being

depleted constantly from modern commercial fishing techniques. Aquaculture, which produces

about half the seafood available in the market today is, could become a major tool for meeting

global fish demands. As the world population increases at exponential rates and wild fish

populations decrease, it is inevitable that the demands for aquaculture farming will increase

rapidly in the future. Aquaculture can also help regenerate the wild seafood stock by providing

a consistent supply of seafood all year round (Rohana Subasinghe, Soto, & Jia, 2009).

21

2- Preserving the population of wild fish species

While preserving the wild fish stock in the oceans around the world, it would also preserve

other marine species that are harmed by overfishing (Weir & Grant, 2005).

3- More benefits with less resources

Aquaculture requires less land, water and other resources compared to other forms of livestock

farming. Another major benefit of fish farming is that fish are cold blooded, and need little

attention in the winter. Because they do not need additional energy to cope with the weather,

there is more output from less input. In other words, energy is conserved. (Naylor, Goldburg,

Primavera, & Kautsky, 2000).

2.6 Antimicrobials

2.6.1 Definition of antimicrobials

Antimicrobials are substances that have the ability to kill or inhibit the development of

microorganisms. Antibiotics can be gotten from natural sources or have synthetic origins.

Antibiotics should be safe (non- poisonous) to the host, permitting their utilization as

chemotherapeutic agents for the treatment of bacterial infectious sicknesses (Burridge, Weis,

Cabello, Pizarro, & Bostick, 2010).

2.6.2 Antimicrobials use in fish culture

Antimicrobials are necessary for the treatment and avoidance of irresistible illnesses in human

and to protect farm creatures including fish. All antimicrobial agents utilized as a part of

veterinary pharmaceutical are the same or firmly identified with antibacterials utilized as a part

of human prescription.

The tremendous increase in aquaculture production has been associated with the use of large

amounts of veterinary drugs to maintain the health of animals, prevention and treatment of

disease outbreaks, and magnify products (Romero et al., 2012).

In aquaculture, illnesses outbreaks are known to be a major constraint to the improvement of

this strip, with a global assessment of disease losses in the range of several billion US$ per

year (Subasinghe, Arthur, Phillips, & Reantoso, 2000).

23

This is especially true for intensive culture system where fish are raised at very high densities

and the risk of disease outbreaks is high because the organism is continuously stressed. To

reduce diseases outbreak in aquaculture, a wide range of medication are used including

vaccines, antibiotics, antiparasitics, antifungal agents, and immunostimulants (Wardle and

Boetner, 2012). The use of these products, has made it possible to accomplish awesome

advances in aquaculture generation limit and to accomplish manageable creation (Fernandes,

Lalitha, & Rao, 1991)

As is the case in terrestrial livestock production, antimicrobials, including antibiotics, have been

widely used worldwide in aquaculture. There are three essential ways, in which antimicrobials

are utilized as a part of aquaculture: 1) therapeutically such as oxytetracycline, to treat existing

infectious disease caused by a variety of bacterial pathogens of fish including Aeromonks

hydrophila, Aeromonas salmonicida, Edwardsiella tarda Pasteurella piscicida, Vibrio

anguillarum, and Yersinia ruckeri., 2) prophylactically, at subtherapeutic concentrations, such

as; oxytetracycline, althrocin, ampicillin, sparfloxacin, and enrofloxacin which are used in fish

farms for prophylaxis and 3) subtherapeutically, for growth promotion, such as those of

ciprofloxacin, enrofloxacin, and other drugs which are used to improve larval survival in

hatcheries.

2.6.3 Antibiotics administration route and fate

In aquaculture, antibiotics are generally administered in feeds, sometimes as a bath and are

occasionally injected. The most well known route for the arrival of antibiotics to fish occurs

through blinding the antibiotic with specially formulated fish feed which is then put in the water

where the fish are kept. Antibiotics are either added during feed manufacture or surface-coated

onto pellets by the manufacturer or the farmer. Many antibiotics are stable chemical compounds

that are not effectively metabolized by fish and remain active where they largely pass into the

environment after being excreted in feces and urine. It has been estimated that 75 percent of the

antibiotics fed to fish are excreted into the water (Burridge et al., 2010). A considerable amount

of antibiotics will be also loss to environment via undigested waste feed falling and

accumulating in the sediments at the bottom of the farm (Burridge et al., 2010).

Undigested feed fragments and fecal matter containing the antibiotics may be reach the marine

environment where they either ingested by wild organisms including fish and filter-feeding

organisms such as mussels and oysters; or transported to the sediment where they accumulated,

or they dispersed by the marine currents (Coyne, Smith, & Moriarty, 2001).

26

Sometimes, antimicrobials may be dispersed into the water in fish farms as a bath. In this case,

the unabsorbed antibacterials will release to the surrounding environment via the effluent.

Occasionally, antibiotics are injected into fish directly.

2.6.4 Harmful effects of antimicrobials use

It is well recognized that the widespread use of antimicrobials in food animal production has

led to the emergence and spread of antimicrobial resistant bacteria and resistance genes, and

the occurrence of antimicrobial residues in products of animals. Resistant bacteria and antibiotic

residues have been detected in living chickens, bovines, and fish as well as in related food

products (Baquero, Martínez, & Cantón, 2008).

1.4.6.2 Mechanisms of development of antibiotic resistance

Due to the over and wrong utilization of antimicrobials, there has been a continuous rise in

numbers of antibiotic–resistant bacteria, which represent a worldwide public health issue. A

resistant microorganism is one which is not killed by an antimicrobial agent after a standard

course of treatment (Levy & Marshall, 2004).

Antimicrobials used to battle disease forces bacteria to either adjust or killed regardless of the

dose or time traverse. The surviving microorganism carry the drug resistance gene, which

would then be able to be transferred either within the species/genus or to other unrelated species.

Clinical resistance is a complex phenomenon and its manifestation is dependent on the type of

bacterium, the site of infection, distribution of antimicrobials in the body, concentration of the

antimicrobials at the site of infection and the immune status of the patient (Pana, 2012).

2.6.4.2 Risks associated with the use of antimicrobials in aquaculture

The use of large amounts of a variety of antibiotics in aquaculture may poses serious risks to

human and fish health through producing of fish and human pathogens that are resistant to

antibiotics or through producing environmental bacteria resistant to antibiotics . It may also

poses risk of exposing non-target animals that might serve as food for humans to antibiotics.

Though still uncertain, there is also concern for the long-term environmental impacts of using

antibiotics in aquaculture (Romero et al., 2012).

2.6.5 Effects of antibiotic on public health

Excessive uncontrolled antibiotic use in aquaculture may cause many risks to human health. It

has been reported that the unconscious use may be the causative agent of antimicrobial resistant

25

human pathogenic bacteria and cause public health problems (Romero et al., 2012), proposing

that antimicrobial resistance genes may be transferred from fish to human pathogens and this

may occur through direct consumption of fish or fish product contain antimicrobial-resistant

bacteria. And the human pathogen subsequently turn to a resistant bacteria to antibiotic encoded

on the transferred-resistance genes, like the resistance gene R Plasmid- harboring which have

been transferred from the fish pathogen A. salmonicida to a human pathogenic E. coli strain

(Kruse & Sørum, 1994).

With the increasing human consumption of products of aquaculture, the probability of

exposure to potentially occurring antimicrobial resistant bacteria, resistance genes or

antimicrobial residues in seafood is increasing. This probability increases if seafood is

consumed with little or no heat treatment (WHO, 2006).

In addition, the gradually raised interest in fish farming production and increasing the

consumption of its products this would increase the human exposure to resistant bacteria and

resistance genes in fish farms and products of aquaculture through professional activities or

food handling. This way of exposure also shows a continuous route for spread of resistant

bacteria and resistance genes from fish farms products to humans.

The human health subsequently facing a big problem in treatment of antimicrobial resistant

bacteria including an increased severity of infection and raising the number of failed treatments,

which can lead to longer diseases period, increased chances of bloodstream infections, and

higher death opportunity (WHO, 2006).

The use of antimicrobial agents by humans may cause disturbance to the normal flora in the

intestinal tract, placing these individuals at increased risk of some infections. Therefore,

individuals who take antimicrobial agents for any reason are more likely to be infected with

pathogens resistant antimicrobial agents.

High-risk populations include individuals working in aquaculture facilities, populations living

around aquaculture facilities, and consumers who regularly eat aquaculture products (Sapkota

et al., 2008).

24

2.6.6 Adverse environmental impacts

About 70-80% of drugs used in aquaculture lastly excreted in the environment. This pollution

with antimicrobials lead to resistant in non-pathogenic microflora such as those found within

the gastrointestinal tract of fish and in the local environment (Kalyva, 2017).

The greatest risk related to antimicrobial use in fish farms is thought to be the advancement of

a reservoir of mobile resistance genes in bacteria in water environments. Such genes can be

disseminated by horizontal gene transfer to other bacteria and ultimately reach fish human

pathogens with which they might come in contact, and thereby potentially cause treatment

problems due to resistance (Grigorakis & Rigos, 2011).

For example, the prevalence level of resistant bacteria to oxolinic acid and oxytetracycline

(OTC) utilized on particular fish aquacultures was higher in the intestinal content of fish

samples taken at the farm after medication than in samples gathered before medication or from

untreated regions (Ervik, Thorsen, Eriksen, Lunestad, & Samuelsen, 1994).

In another study, a significant rise in frequency of bacteria resistant to antimicrobial agents used

on specific fish farms at Galway Bay have been detected from sediments under a marine fish

cages received 175 kg oxytetracycline over 12 days (Kerry et al., 1994).

Similar results have been found upon treatment of salmonids at fish farms located in Puget

Sound, Washington with various antibiotics (including oxytetracycline); significant increase in

the proportion of antibacterial-resistant bacteria was found in the sediments of farm used the

greatest amount of antibacterials. In comparison, in farm used the least amount of antibacterials,

the percentage of sedimentary bacteria that were resistant to the antibacterials were lower

(Herwig, Gray, & Weston, 1997).

In western Denmark, in comparing the proportions of antibiotic-resistant microflora from inlet

and outlet samples, it was found an increase in the proportions of antibiotic-resistant and high

levels of individual and multiple antimicrobial resistances within bacteria in outlet samples

compared to that of inlet samples, indicating a substantial impact of fish farming on bacteria

associated with aquacultural environments (Schmidt, Bruun, Dalsgaard, Pedersen, & Larsen,

2000).

27

2.6.7 Exposing other (non-target) animals that may act as food for humans to antibiotics

Antibiotics may end up in non-target creatures associated with fish culture sites. Wild fish,

crustaceans, and mollusks living in the region of marine fish aquaculture have been appeared

to aggregate quantifiable levels of anti-microbials in their tissues because of nourishing on

waste medicated feed and feces. It is possible that antimicrobial residues in non-target animals

might represent a pathway by which antibiotics could enter human populations (Samuelsen,

Torsvik, & Ervik, 1992). Björklund, Bondestam, and Bylund (1990) detected residues of

oxytetracycline in the wild bleak fish (Aburnus alburnus) samples obtained from a location near

a salmon farm in Norway. In this study, bacteria, resistant to OTC, were also isolated from the

intestines of wild fish.

In another study conducted in Puget Sound, Washington, samples of oysters (Crassostrea gigas)

and Dungeness crabs (Cancer magister) and red rock crabs (Cancer productus) were taken

from the vicinity of, and under, a salmon farm during, and within 12 days of, an OTC treatment.

About half of the sampled red rock crabs contained OTC at levels ranging from 0.8µg/g to at

least 3.8 µg/g muscle (Capone, Weston, Miller, & Shoemaker, 1996).

In Norway, (Samuelsen, Lunestad, Husevåg, Hølleland, & Ervik, 1992) sampled wild fishes in

the vicinity of two Norwegian marine salmon farms treated with oxolinic acid. The samples

were obtained on the last day of treatment. A high concentration (12.51 µg/g) of oxolinic acid

was detected in the coalfish, Pollachius virens. In mussels near the farm, oxolinic acid levels

of 0.65 µg/g were found.

2.6.8 Adverse Ecological impacts

Antimicrobials released into the marine environment can accumulate in sediments where they

persist there for long period affecting aquatic organisms. In contrast, given the rapid dilution

and susceptibility of many drugs to photodegradation, antimicrobials pass to surrounding water

are not a major concern.

Because of the antibiotic toxicity to microorganisms, they may affect the composition of the

phytoplankton and the zooplankton communities and even the diversity of populations of larger

animals. In this manner, potential alterations of the diversity of the marine microbiota produced

by antibiotics may alter the natural balance of the marine environment and affect complex forms

of life including fish, shellfish, marine mammals, and human beings (Burridge et al., 2010).

28

The presence of large amounts of antibiotics in the water and sediment can affect the flora and

plankton in culture systems, causing shifts in the diversity of the microbial communities and

affecting the structure and activity of microbiota (Hunter-Cevera, Karl, & Buckley, 2005).

Plankton community composition were affected by antibiotics, with toxicity varying widely

depending on application rates and natural factors ((Isidori et al., 2005); (Christensen,

Ingerslev, & Baun, 2006). Accumulation of antibiotics in sediments may interfere with bacterial

communities and affect the rate and mechanism for mineralisation of organic wastes and the

processes of nitrogen fixation (McLean, 1997). The heavy use of antibiotics inhibits the

microbiota at the base trophic level in the water and sediment from performing important

metabolic functions, inducing algal blooms and anoxia that could potentially lead to fish kills

and impacts on human health (Cabello, 2006).

2.7 Antimicrobial residues

The extensive use of antimicrobial for the treatment of bacterial diseases in aquaculture can

result in residues of antimicrobials in the food product. Several classes of antibiotics are

commonly used in large quantity in fish industry, especially in developing countries where their

uses are not regulated. Some of these antibiotics are often non-biodegradable and deposit in the

edible tissues (fish meat) offered for human consumption as antimicrobial residues (Johnson,

2014).

2.7.1 Definitions

The term "drug residues" is used to describe the amount of the medication that can be

distinguished in tissues at determined circumstances after administration of the drug have been

stopped (Burgat-Sacaze, Delatour, & Rico, 1981).

Maximum residue limit means the highest amount of residue resulting from the use of a drug

product, which may be legally authorized or recognized as acceptable in or on a food, specify

to individual food goods. It is based on the type and concentration of residue considered to be

without any toxicological hazard for human health as expressed by the Allowed Daily Intake

(ADI), or on the basis of a temporary ADI that utilizes an additional safety factor (Myllyniemi,

2004).

2.7.2 Harmful effects of antimicrobial residues

The public health problems associated with antimicrobial residues relay on the amount of the

antimicrobial consumption, i.e. the exposure. In general, the higher the exposure, the higher the

29

risk. Residues may produce acute or cumulative allergic, toxic, mutagenic, teratogenic or

carcinogenic effects in susceptible individuals who eat meat that contains antibiotic residues.

For example, chloramphenicol, the broad-spectrum antimicrobial agent, that used in

aquaculture as a prophylactic agent against carp dropsy (caused by Aureobacterium

liquefaciens) and has been used in the treatment of trout ulcer disease (caused by Haemophilus

piscium) and furunculosis (caused by A. salmonicida), presents a specific danger to human

wellbeing as it can cause an idiosynchratic dose-independent aplastic anemia in humans, which

can be induced by low concentrations of chloramphenicol ((Settepani, 1984); (Sundlof, Cooper,

& Miller, 1997)).

Penicillin, the most commonly implicated antimicrobial in adverse reactions from foodborne

residues, causing penicillin hypersensitivity or skin disease unrelated to penicillin allergy.

Penicillin residues as low as 5-10 IU are capable of producing allergic reactions in previously

sensitized persons (Dayan, 1993). The extent of use of penicillin in aquaculture is most probably

low due to the fact that penicillin rapidly degrades in aqueous solutions.

Many antimicrobials other than penicillin – including other beta-lactams, streptomycin (and

other aminoglycosides), sulfonamides and, to a lesser extent, tetracyclines - are known to cause

allergic reactions in sensitive persons. However, apart from a single report of a reaction to meat

suspected of containing streptomycin residues, we are not aware of any reports of foodborne

allergic reactions resulting from residues of any antimicrobial other than penicillin (WHO,

2006).

Increasing concern about the carcinogenic and mutagenic potential and their thyroid toxicity

has led to decreased use of sulfonamides. Research has shown that chronic dietary exposure to

sulfamethazine produces a statistically significant increase in thyroid follicular cell adenomas

in both rats and mice, and a statistically significant increase in thyroid follicular cell

adenocarcinomas in rats (WHO, 2006).

Antibiotic residues transferred to humans through food can also affect human health by altering

the intestinal microflora through the emergence of resistant strains to frequently used antibiotics

and promoting the development of acquired resistance in pathogenic enteric bacteria (Marshall

& Levy, 2011).

2.7.3 Factors contribute to the drug residue problem

Many factors lead to the drug residue issue. In many countries, there is a lack of instruction for

good drug use. In other countries with labeling, Failure to adhere to withdrawal times is the

most usual reason, while use of an unapproved drug, and administration of increased dosages,

11

also results in violative residues In most countries where antimicrobials are licensed for use in

aquaculture, withdrawal times that ensure safety to the consumers are set. Non-compliance with

these withdrawal times presents a risk to public health (WHO, 2006).

Most countries have implemented monitoring or surveillance programs in regard to drug

residues in food animals, including farmed fish. In most countries, chloramphenicol has been

banned for use in food producing animals, including aquaculture, because of the risk of severe

human disease associated with chloramphenicol residues. An acceptable daily intake (ADI) has

never been allocated for chloramphenicol and, consequently, a maximum residue limit (MRL)

has not been assigned (WHO, 2006).

This has resulted in the restriction of its use in veterinary medicine to non-food use. Despite

these restrictions, chloramphenicol has been detected in national monitoring programs during

the past years and these residues have caused safety concerns. Shrimps, prawns and food

products from aquatic animals were among the commodities in which the drug was detected

(WHO, 2006).

2.7.4 Screening technique for the detection of antimicrobial residues in food products

A screening method is known as the first process that is used to sample analyses. The objective

is to emphasize the presence or absence of drugs residues. It should be as simple as possible.

As well as, it may be rather complex, due to, e.g. the properties of the drugs of interest or the

wanted limit of detection, and in some cases, will provide (semi) quantitative next to the

qualitative data (Aerts, Hogenboom, & Brinkman, 1995).

2.7.4.1 Classification of screening methods by detection principle

1. Biological methods: reveal cellular responses (e.g. inhibition of bacterial growth) to

analytes. These methods are not selective and can include several chemical classes of active

analytes (e.g., antimicrobials). They do not primates the identification of single analytes.

2. Biochemical methods: detect molecular interactions (e.g. antigens, proteins) between

analytes and antibodies or receptor proteins (e.g. ELISA), chemical labeling of either the

analyte or antibody/receptor allows the interaction to be monitored and measured. These

methods are either selective for a family of analytes having related molecular structures or are

sometimes analyte specific.

3. Physicochemical methods: distinguish the chemical structure and molecular characteristics

of analytes by separation of molecules (e.g. TLC, GC, HPLC) and the detection of signals

12

related to molecular characteristics (e.g. UV, DAD, ..etc). They are able to distinguish between

similar molecular structures and allow the simultaneous analysis of several analytes.

Table (2.1): Demonstrates advantages and disadvantages of different screening methods of

residues analysis (Sirdar, 2011).

Test Advantages Disadvantages

ELISA Ease of use

Availability for a good number of specific

compounds.

Availability for families of compounds (e.g.

sulfanomides, estilbenes).

Large number of samples (42) per kit for a

single analyte.

Reduced time to obtain the results (2-2.5 h for

most kits).

High sensitivity and specificity.

Possibility to use within the food processing

facility.

Expensive

Limited storage (few months)

under refrigeration.

The need for waste disposal.

Interferences giving some false

positives.

Only one kit per residue

searched.

Biochip array

biosensors

Easy to use.

Results available in short time.

Multiples residues analyzed in one shot (as

many as in an array).

Full automation: higher productivity.

High through-put technique: up to 120

samples per hour and array.

High operative costs chips and

equipment cost.

Analysis restricted to available

chips

HPLC Reduced time (few hours) to obtain results.

Sensitive

Automation leading to higher productivity.

Specificity depending on a detector

Expertise required.

Needs sample preparation

(Extraction, filtration, addition

of internal standards, etc.).

Expensive.

Microbial

methods

Can be used for large surveillance

programmers.

Basic laboratory equipment.

Broad spectrum.

Easy to use.

Economical.

Difficult to standardize

preparation procedures.

Some test could not insure

MRLs compliance.

Sample preparation required to

remove false positives due to

protein bacterial inhibitors.

Low sensitivity.

11

Determination of antimicrobial residues in food products such as meat, milk, and eggs by

microbiological methods depends on the effect on a specific microorganism, the spectrum and

the mode of action of the antimicrobials which will be determined. On the other side residue

determination by chemical methods such as chromatography (by all its types) depends on the

chemical properties (Mitchell, Griffiths, McEwen, McNab, & Yee, 1998).

2.7.5 Confirmation methods

Confirmation methods can be both qualitative and quantitative. Many confirmation methods

have been described for the detection of veterinary drugs in various matrices. These techniques

comprise liquid chromatography (LC), Gas chromatography (GC) and mass spectrometry

(Tothill, 2003).

2.7.6 Microbiological inhibition tests

Microbiological inhibition tests for AMR exploit the property of these compounds and their

selective toxicity towards specific bacteria. Some inhibition tests use growth medium

inoculated with bacterial spores and a pH or redox indicator as an indicator for growth. In the

absence of AMR, or if the concentrations are below the detection limits, the test bacterium will

start to grow, producing acid compounds that change the indicator color, permitting visual or

photometric detection. Nevertheless, if an antimicrobial is present in the sample no color change

is observed (Pikkemaat, 2009). In other tests, called agar diffusion tests, the growth medium is

inoculated with a specific bacterial test organism and samples are applied on top of agar media,

or in a well in the agar layer. After over-night incubation, the presence of an antimicrobial

residue becomes visible as an inhibition zone around the sample.

Bacterial inhibition tests used to screen tissues for antimicrobial activity include the Swab Test

on Premises (STOP), the Calf Antibiotic and Sulfa Test (CAST), the Fast Antibiotic Screen

Test (FAST), the Charm Farm Test (CFT), the Antimicrobial Inhibition Monitor 96 (AIM-96)

assay, the German Three Plate Test, the European Union Four Plate Test (FPT) and the New

Dutch Kidney Test ((Korsrud, Boison, Nouws, & MacNeil, 1997);(Okerman et al., 2004)). The

size of the inhibition zone depends on the type of residue and its concentration, while the

sensitivity of the tests (the detection limits of antibiotics) are affected by many factors, such as

indicator organism, pH, type of growth medium, and thickness of the agar layer (Bovee &

Pikkemaat, 2009). For example, the pH of the test medium may influence the detection limits

of most antibiotics (Korsrud et al., 1997).

13

2.7.7 Examples of microbiological assay methods

A comparison of some microbiological inhibition tests for AMR are summarized in Table 2.2

(Myllyniemi, 2004).

Table (2.2): Agar diffusion tests used for the screening of antimicrobial residues in meat.

Reference/producer Test

matrix

Additional

substances

Medium

PH

Test medium Test bacterium Test method

Bogaerts and Wolf.

1980

Muscle

Kidney,

liver

Trimethoprim

(PH7.2)

6.0,7.2,

8.0

Test agar B.subilis BGA,

M. luteus ATCC

9341

Four plate

test (FPT)

USDA,

1979

Johnston

Et all …. 1981

Kidney

Tissue

fluid

Kidney,

liver

7.9 Antibiotic

Medium no 5

B.subtilis Swab test

on premises

(STOP)

MAF,

2001a

Kidney,

muscle

Trimethoprim

(Ph 8.0)

6.0,8.0 Test agar B.subtilis BGA Two _plate

test

USDA 1983 Urine

from live

animals

7.9 Antibiotic

Medium no 5

B.subtilis

ATCC 6633 The live

animal

swab test

(last)

Nouws et all …

1988

Renal

pelvis

fluid

Dextrose

phosphate

buffers,

trimethoprim

7.0 Standard

nutrient agar

B.subtilis BGA New dutch

kidney test

(NDKT)

Koenen- dierick at

al….1995

Kidney,

Muscle

Dextrose

trimethoprim

7.0 Standard

nutrient agar

B.subtilis BGA Belgian

kidney test

(BKT)

USDA,

1984

Kidney

tissue

fluid,

Muscle,

liver

Dextrose

Bromcresol

purple

7.4 Mueller_Hinton

agar

B.megaterium Calf

anbbiotic

and sulfa

test (CAST)

USDA,

1994

Kidney

Tissue

fluid

7.4 Mueller_Hinton

agar

B.megaterium The fast

anbbiotic

screen test

(FAST)

DSM Food

Specialities

Muscle,

Kidney,

liver,

Urine

B.

Stearothermophilus Premi test

16

2.7.8 Other tests

Specified products or groups of related antibiotics can be detected with solid-phase

fluorescence immunoassays (SPFIA) or with receptor tests. A combination of four SPFIA tests,

for example, enables the detection in one run of: (1) the group of the tetracyclines, (2) ceftiofur

and cefquinome, (3) penicillin, ampicillin and amoxicillin, and (4) cephapirin, in milk or in

kidney tissue (Lieve Okerman, Wasch, Hoof, & Smedts, 2003).

2.8 Residue Control Programs

Residue control programs are designed in accordance with country regulations. These programs

generally control both domestic and imported products. Drugs included in these programs are

selected on the basis of their risk profiles (Cunningham, Elliott, & Lees, 2010). Control

programs have two principal components: monitoring and surveillance. Residue monitoring

program randomly collect sample tissues from animals then tissue samples are screened for

residues. The residues are then assessed for compliance with the applicable MRL. Surveillance

programs, on the other hand, collect sample tissues from animals suspected of violative residues

depending on clinical signs or herd history. If monitoring reveals a potential residue problem,

the action taken will vary in accordance with country rules (Paige, Chaudry, & Pell, 1999).

2.9 Withdrawal period

To insure that drug residues have declined to a safe concentration following the use of drugs in

animals, a specified period of drug withdrawal must be observed prior to providing any products

for human consumption. It is the time which passes between the last dose given to the animal

and the time when the concentration of residues in the tissues: muscle, liver, kidney, skin/fat or

products milk, eggs, honey is lower than or equal to the maximum residue limit (Cholas, 1976;

Nouws & Ziv, 1978).

2.10 Previous studies for antibiotic residues

Abu Bakar, Ayub, Muhd Yatim, and Abdullah Sani (2010), investigate the presence of

pesticide and antibiotic residues in freshwater farmed fish, and results shows that just 2.9% of

fish contain pesticide residues and 5.8% contain antibiotic residues.

Antibiotic residuals were assessed in some farmed rainbow trout and the obtained results

showed that the residual of these antibiotics in trout muscles of some fish farms were higher

15

than the acceptable levels and therefore, requires a serious attention of both the environment

and the consumer health care (Soltani et al., 2014).

In Iran, the antibiotic residues were determined in farmed rainbow trout using HPLC analysis

and Oxytetracycline, tetracycline, enrofloxacin, ciprofloxacin, and florfenicol residues were

detected in 6.76%, 37.8%, 31.1%, 10.8%, and 14.9% of the samples, respectively; while

chlortetracycline was not detected (Barani & Fallah, 2015).

The presence of antimicrobial residue in catfish sold for human consumption in Ibadan

metropolis, Nigeria was determined based on the inhibition of growth of Bacillus

stearothermophilus. The results showed that appreciable quantity of catfish consumed in

Ibadan, posed antibiotic residue risks and food safety consequences (Olatoye & Basiru, 2013).

2.11 Fish Microbial quality

In their aqueous environments, fish are exposed directly to a wide variety of microorganisms

through surface contact. After harvesting, bacteria may be also transferred to the fish during

careless handling of landed fish by persons serving in food processing industries, its stowing,

cutting and storage. A number of microorganisms including Staphylococcus aureus have been

isolated from the hands of employees working in food establishments (Pal & Mahendra, 2015).

Live and newly caught fish carry their microbial load on the slime layer on the surface of the

skin, in the gastrointestinal tract and in the gills.

Bacterial species exist in fish species as normal flora and pathogenic forms. the majority of

Normal flora of fish is Gram negative genera including: Acinetobacter, Flavobacterium,

Moraxella, Shewanella and Pseudomonas. Members of the families Vibrionaceae (Vibrio and

Photobacterium) and the Aeromonadaceae (Aeromonas spp.) are also common aquatic bacteria,

and typical of the fish flora. Gram-positive organisms such as Bacillus, Micrococcus,

Clostridium, Lactobacillus and coryneforms can also be found in varying proportions (Huss,

1995). It is suggested that, fish microflora tends to reflect the microbial communities of the

surrounding waters (Rhea, 2009).

The pathogenic bacteria associated with fish and fishery product have been categorized as

indigenous and non-indigenous (Kvenberg, 1991). The indigenous bacterial pathogens are

found as normal flora in the fish’s habitat such as the pathogenic Vibrio species, Aeromonas

species (Petronillah R Sichewo, Gono, & Sizanobuhle, 2013), and Plesiomonas shigelloides.

14

On the other hand, the non-indigenous are introduced to fish as a result of habitat contamination

(mainly fecal contamination and includes Salmonella spp., Shigella dysenteriae, pathogenic

Escherichia coli, Staphylococcus aureus) or during harvest, processing, storage or preparation

for consumption (examples include Bacillus cereus, Listeria monocytogenes, Staphylococcus

aureus, Clostridium botulinum, Clostridium perfringens, Salmonella spp. and Yersinia

enterocolitica).

In normal situation, bacteria seldom cause any problem, as the fish’s own immune system is

capable of fending off any infection. They however, become pathogens when fish are

physiologically unbalanced, nutritionally deficient, or there are other stressors, i.e., poor water

quality, overstocking, which allow opportunistic bacterial infections to prevail (Austin, 2011).

Evidence indicates that the number and diversity of microbes associated with fish is highly

variable and depend on the geographical location, the season (Strunjak-Perovic, Kozacinski,

Jadan, & Brlek-Gorski, 2010) and environmental factors around it.

Fishes live in fresh waters, or in salt water (sea and oceans), and the types of bacteria vary

depending on the type of water in which fish are present.

The bacteria present in or on fish may also influenced by the harvesting method and subsequent

processing. In Gaza strip for example, the fishing gears employed in catching fishes may affect

the microbiological quality of the fish that is brought to the markets. Hook and hand cast net

fishing for instance keeps the earlier caught fish waiting, often without chill storage in ice while

the fisherman tries to catch more fish to trade. In net fishing such as beach seine net, gillnets

and entangling nets, the nets are laid in water for several hours. In such cases, if fish dies under

water, the high ambient temperatures as is the case in Gaza strip (average surface temperature

in eastern Mediterranean basin over 21oC), will enhance the microbial growth on fish soon

under water. In Gaza strip, there is also lack of investments in landing sites, handling and selling

sites, resulting in poor sanitation and hygiene. After catching, fishermen sell their products in

open fish markets or in streets by vendors which may contribute to more contamination and

multiplication of microorganisms and hence poor quality of fish are presented to the consumers.

Fresh and lightly preserved fish are susceptible to spoilage soon after death. Bacteria colonizing

the skin, gills and intestines began to replicate rapidly utilizing the fish protein and food

nutrients. The rate of degradation during spoilage depends on the microorganisms associated

with aquatic environments, the initial microbial load, ambient temperature and improper post-

harvest handling. Discoloration, dark-red gills, sunken eyes slime formation, changes in texture,

strong odors, off –flavors, and gas production are some signs of spoiled fish. Many spoilage

17

causing microorganism including bacteria (Aeromonas, Alcaligenes, Bacillus, Enterobacter,

Enterococcus, Escherichia coli, Lactobacillus, Listeria, Pseudomonas, Shewanella) and fungi

(Aspergillus, Candida, Cryptococcus, Rhodotorula) were isolated from fresh and spoiled fish

and other sea foods (Pal,2012).

Spoilage renders fish or fish products less acceptable, unacceptable or unsafe for human

consumption (Pal, 2012), constituting therefore, an economic loss to fishermen and fish traders.

For example, spoilage accounts a loss of 10 to 12 million tons per year and 20 million tons of

fish in a year are discarded at sea (Kumolu-Johnson & Ndimele, 2011).

Previous studies have demonstrated the presence of indicator microorganisms of fecal pollution

(Petronillah R Sichewo et al., 2013), opportunistic and pathogenic bacteria to humans in fish.

Microbes that live on fish affect the quality of fish and can cause diseases for humans, especially

when they are opportunistic and or pathogenic in nature (Petronillah Rudo Sichewo, Gono,

Muzondiwa, & Mungwadzi, 2014).

It has been reported that fish consumption can be a good source for human pathogenic bacteria

and other food borne diseases exposure to human (Pires et al., 2009). Pathogenic bacterial

species form fish can be inherited to humans and might cause food borne infections such as,

dysentery, typhoid, fever, diarrhea, salmonellosis and cholera. The transmission of these

microbes to humans can be through improperly cooked food or the handling of the fish.

It was reported that harmful microorganism could also enter seafood processing chain because

of inadequate process control, poor standards of hygiene and sanitation in processing plants and

post-production contamination during incorrect handling or storage.

Fishery products have been recognized as a potential source of food-borne pathogens (Yücel &

BALCI, 2010).

The consequences of poor microbiological fish quality are far reaching. Great economic losses

due to treatment expenses have been reported from foodborne illness as the result of consuming

contaminated fish. Such diseases can be a trouble to the immune compromised, children and

elderly people.

This study reports the microbiological quality in the edible portion of frozen fish brought from

local markets, farmed fish and open marketed fish caught from the Gaza shore. Muscles with

(or without) skin represent the edible portions of fish by Palestinian people. Although, muscles

of healthy, freshly caught fish are assumed to be sterile (Pamuk, Gurler, Yildirim, & Siriken,

2011), the skin may carry substantial numbers of bacteria.

18

There is paucity of information on quality of fish sold in Gaza strip. Accordingly, the present

study was undertaken to investigate some microbiological indicators in the edible parts of five

fish species marketed in the different markets in Gaza strip to assess the microbiological quality

of fish in order to give an impression of the hygienic quality of the fish sold in Gaza strip

For farmed fish, it is essential to investigate the microbial indicators associated with grow-out

culture in order to develop safe farm management practices for the production of fish safe for

human consumption and for prevention of fish diseases. This study also evaluated the

microbiological quality of rearing water of farmed fish, as a potential factor correlated to

bacterial population associated with farmed fish. The information obtained should allow a better

control of the bacteriological parameters in the culture water

2.12 Microbial indicators

Indicator organisms may be utilized to mirror the microbial quality of foods relative to product

shelf life or their clearance from pathogenic bacteria. In general, indicators are most often used

to assess food safety/sanitation (Jay, Loessner, & Golden, 2005). Fish quality is mainly assessed

through the total aerobic plate counts and counts of bacteria with public health relevance such

as total coliform,S. aureus, salmonella and Vibrio spp. Monitoring of these microorganisms

have been suggested as a measure of fish quality.

2.12.1 Staphylococcus aureus

S. aureus is a facultative anaerobic Gram-positive coccus; it is non-motile, non- spore forming

and catalase and coagulase positive. Cells are spherical single or paired cocci, or form grape-

like clusters. Its cell wall is resistant to lysozyme and sensitive to lysostaphin, which specifically

cleaves the pentaglycin bridges of Staphylococcus spp. Some S. aureus strains are able to

produce staphylococcal enterotoxins (SEs) and are the causative agents of staphylococcal food

poisonings. S. aureus is remains a major cause of Food-borne diseases (FBDs) because of its

contamination of food products during preparation and processing (Le Loir, Baron, & Gautier,

2003).

S. aureus has the ability to grow in a wide range of temperatures (7° to 48.5°C with an optimum

of 30 to 37°C), pH (4.2 to 9.3, with an optimum of 7 to 7.5) and sodium chloride concentrations

(up to 15% NaCl). These capabilities enable S. aureus to grow in a wide sort of foods (Le Loir

et al., 2003).

19

S. aureus is an important pathogenic bacterium of humans and animals. It causes a many types

of diseases, ranging from relatively harmless localized skin infections to life-threatening

systemic conditions (Bukowski, Wladyka, & Dubin, 2010).

Staphylococcus auerus may possibly contaminate seafood products as a result of poor handling

which might eventually affect the health of consumers (Okonko et al., 2008).

2.12.2 Salmonella spp.

Salmonella is a flagellated, rod shaped, Gram-negative facultative anaerobic, non-endospore-

forming bacterium, facultative intracellular, a member of Enterobacteriaceae family, trivially

known as "enteric" bacteria (Ray & Bhunia, 2007). Salmonella are intracellular pathogens in

cold-blooded as well as warm-blooded animals and important zoonotic agents (Jacobsen,

Hendriksen, Aaresturp, Ussery, & Friis, 2011).

The genus Salmonella includes two species Salmonella enterica and Salmonella bongori, with

S. enterica being divided into six subgroups (enterica, salamae, arizonae, diarizonae, indica,

and houtenae) (Pignato, Giammanco, Santangelo, & Giammanco, 1998).

Salmonella are a major cause of food-borne disease throughout the world. Implicated foods are

typically beef, pork, poultry, dairy products, but also eggs and fresh produce. Salmonella typhi

and paratyphi A can be spread by eating food that has been improperly handled by infected

individuals, or by drinking water that has been contaminated by sewage containing the bacteria.

The incidence of Salmonella infections due to seafood consumption is low compared with

salmonellosis associated with other foods. However, detection of Salmonella spp. in seafood

can not be ignored as it is responsible for most of the foodborne diseases or gastroenteritis

characterized by diarrhea, abdominal cramp, vomiting, nausea, and fever. According to Centers

for Disease Control and Prevention, Salmonella is the leading cause of bacterial foodborne

illness causing approximately 1.4 million nontyphoidal illnesses, 15,000 hospitalizations, and

400 deaths in the USA annually (Newell et al., 2010).

2.12.3 Total plate count

The study also reports on total plate count, organisms of public health significance for quality. Total

Plate Count, the heterotrophic plate count (HPC) is an assessment of the number of bacteria in

a sample that derive their energy from complex carbohydrates and sugars. These bacteria are

common in the environment and can be isolated from soil, surface waters, groundwater, and

31

vegetation. Some of these bacteria such as Pseudomonas spp. are opportunistic pathogens but

may not necessarily be transmitted in drinking water (Kloos & Bannerman, 1994).

2.12.4 Coliform bacteria

Coliforms are Gram-negative, rods facultative anaerobic bacteria. recognition properties used

are production of gas from glucose (and other sugars) and fermentation of lactose to acid and

gas within 48 h at 35ºC (Hitchins, Feng, Watkins, Rippey, & Chandler, 1998).

The coliform group includes species from the genera Escherichia, Klebsiella, Enterobacter and

Citrobacter, and includes E. coli. Coliforms were historically used as indicator microorganisms

to serve as a measure of fecal contamination, and thus potentially, of the presence of enteric

pathogens in fresh water. Although some Coliforms are found in the intestinal tract of human,

most are found throughout the environment and have little sanitary significance (Bej, Steffan,

DiCesare, Haff, & Atlas, 1990).

2.12.5 Vibrio spp.

Non-spore former and are motile, usually by a single polar flagellum. Most are oxidase and

catalase positive and glucose fermenter without gas production. They are normal flora in fresh

water and marine environments and some might be pathogenic to humans. Many of the

pathogenic species, with the notable exception of Vibrio cholerae, are adapted to salt or

brackish water habitats and are halophilic to some degree, being unable to grow in the absence

of sodium chloride.

Three important human pathogens species are - V. cholerae, V. parahaemolyticus and V.

vulnificus. All three have the potential to be foodborne, and are most often associated with the

consumption of raw, or undercooked, shellfish. A number of other species have infrequently

been isolated from the stools of people suffering from gastroenteritis and are considered to be

occasional human pathogens. These include V. alginolyticus, V. fluvialis, V. furnissii, V.

hollisae, V. metschnikovii and V. mimicus. These species are not generally regarded as

significant foodborne pathogens (Farmer & Hickam-Brenner, 2003) (Farmer, 2005).

2.12.6 Previous studies for microbial quality

Rajkowski, Hughes, Cassidy, and Wood-Tucker (2013), tested the microbial quality of catfish

nuggets, results reveals that TPC results comply with the standards of International Commission

on Microbiological Specifications Food (ICMSF). In addition, Salmonella spp. Listeria spp.

and enterotoxin negative S. aureus were seen in little fish samples.

32

Microbial quality indicators were examined by Dib (2014), The study found that the fish that

were tested contaminated with fecal coliform and some of pathogenic bacteria.

In Zimbabwe pathogenic bacteria were Isolated and identified in edible fish. S. typhi, P.

aeruginosa, E. coli, S. aureus and E. faecalis were detected in fish and water samples. These

findings may reflect poor microbial quality of fish and water samples in addition to potential

risk to human health (Petronillah R Sichewo et al., 2013).

A study in Bangladesh assess total viable aerobic count and fecal coliforms in fish samples in

addition to the occurrence of certain fish pathogenic bacteria as Salmonella spp. and Vibrio

cholera. TVAC and fecal coliforms count met the standards International Commission of

Microbiological Specification for Food (Sanjee & Karim, 2016).

Microbiological indicators of frozen fish fillet sold in Sulaimani markets were tested. Result

obtained were met the Iraqi standard regulations of microbial count. All tested samples were

negative for the presence of V. cholera (Murad, Khidhir, & Arif, 2013).

In an investigation to decide the microbial nature of frozen fish sold in Uyo Metropolis, the

outcomes demonstrated that the frozen fish tests were vigorously contaminated which might be

because of poor sterile practices utilized by the sellers. This is of public health concern, as these

organisms are known causes of food-borne diseases (Adebayo-Tayo, Odu, Anyamele, Igwiloh,

& Okonko, 2012).

Another research was undertaken to assess the microbiological quality of 109 fish samples. The

results showed that the count of shrimp per pound, thawing weight loss, and pH of these

products ranged from 20 to 400, from 1.94% to 2.38% and from 7.48 to 7.92, respectively

among frozen shrimp products. The mean count of the total viable count (TVC),

Enterobacteriaceae, coliform and S. aureus varied from 4.8 × 103 to 7.7 × 108, from nil to

5.1 × 104, from nil to 4.1 × 103 and from nil to 5.9 × 102, respectively in the aforesaid frozen

shrimp products. The count of TVC in 40 and 9% samples of block broken peeled, and block

peeled headless frozen shrimp products, respectively was higher than the allowable limit

(106 cfu/g) in the Egyptian Standards of Frozen Shrimp Specification. Enterobacteriaceae,

coliform and coagulase positive Staphylococci were detected in some analyzed samples but in

load less than those recommended by the Egyptian Organization for Standardization and

Quality Control (2005). E. coli was not detected in any of analyzed samples of the 7 frozen

shrimp products (Abd-El-Aziz & Moharram, 2016).

31

A Nigerian study was applied on frozen fish found that total viable count (TVC) ranged from

2.0 x 103 to 7.4 x 103 CFU/g, total meet the acceptable limits for frozen fish. The sanitary,

storage and hygienic conditions of the supermarkets were relatively the same (Oramadike,

Ibrahim, & Kolade, 2010).

33

Chapter III

Materials and Methods

36

Chapter III

Materials and Methods

This chapter presents the materials and methods used to accomplish the aims of this study. This

is a cross-sectional analytical study that aimed to determine the presence of antibiotic residues

of four groups of antibiotics (β-lactams, aminoglycosides, macrolides and tetracyclines) in fish

sold in Gaza strip. The knowledge of Palestinian fish farmers had on the purpose and safety of

antibiotics usage in fish farming and how they are used in fish farms was also determined. In

addition, the microbial quality of fish samples were investigated.

3.1 Materials

3.1.1 Equipment

Equipment used in this study are listed in Table (3.1). These were available from the Public

Health Laboratory at the Palestinian Ministry of Health.

Table (3.1): Equipment used in the study

Items Manufacturer, country

Incubator N-Biotek, Korea

Safety cabinet

Freezer Bio-Equip

Balance Sartorius, Germany

Spectrophotometer CharmTeck

Quebec colony counter, with magnifying lens (Anderman, U.)

Laminar flow Hood Hotte de bacteriologe, France

Stomacher AES, laboratoire, France

pH meter Inolab, Germany

Autoclave KSG, Germany

Water Bath Fried Electric, Occupied Palestine

Refrigerator Sanyo, Japan

Vortex mixer DigiSystem, Taiwan

Digital camera Sony China

Hot plate and Magnetic stirrer Dragon lab, China

3.1.2 Microorganisms, media and reagents

All test microorganisms used in this study are ATCC strains. Reagents are of analytical grade.

Media were purchased from HiMedia, India and were prepared according to manufacturer's

recommendation (Table 3.2).

35

Table (3.2): Microorganisms, media and reagents.

Reagent Manufacturer,

country

Bacillus cereus spores ATCC 11778 KWIK-STIK™,

Microbiologics,

USA Kocuria rhizophila cells ATCC 9341a

Staphylococcus epidermidis cells ATCC 12228

Antimicrobials assay media No 4, 8 and 11 HiMedia, India

Nutrient agar media

Sensitivity antibiotic disks; Te (30), P (10), E (15) and N (5).

Penicillinase BD, USA

K2HPO4 Liofilchem, Italy

KH2PO4

Oxidase test Hy.laboratories

Ltd, Palestine

Absolute Ethanol Sigma, USA

The API-20 E test kit bioMerieux, Inc.,

France

Buffered peptone water

HiMedia, India

Salmonella Shigella agar

Selenite F-broth

Xylose lysine deoxycolate agar

Hektoen enteric agar

Muller Hinton agar

Selenite cysteine broth

Rappaport-Vassiliadis medium Difco, U.S.A

3.1.3 Glassware and disposables

The most frequently used glassware and disposables are listed in (Table 3.3).

Table (3.3): Glassware and disposables used in experimental work

Items Manufacturer

Micropipettes and suitable tips. Dragon lab, China

Sterile scalpels Medipharm, China

Sterile bags. Whirlepak, USA

Stainless steel cylinders HiMedia, India

Eppendorf tubes Eppendorf

Erlenmeyer flasks 100,250 and 500 ml. Rasotherm, Germany

Plastic Petri dishes, 90 x 15 mm. Miniplast

Media bottles, 500 ml. Kimax, USA

3.2 Study area

The study was carried out in Gaza strip, south– western Palestine. Gaza strip lies between

latitudes 31.51667° North 34.48333° East. Gaza strip is located southeast the Mediterranean

Sea, bordered by the Occupied Palestinian territories to the east and north, and Egypt to south.

34

The total area is estimated at 365 km2. Its length along the coast is about 45 km and the width

ranges from 5 to 12 Km. and Gaza strip has a two million population. Gaza strip has a semi-

arid Mediterranean climate, with mean temperature varying from 12-14 oC in January, to 26-

28 oC in August.

3.3 Fish collection

A total of 100 fish samples were investigated in this study. Fish samples were categorized into

three groups; frozen, farmed and caught fish. Group 1 comprised of thirty (30) frozen fish [10

Sutchi catfish fillet (Pangasius hypophthalamus), 10 Argentine hake (Merluccius hubbsi, 10

croaker fish (Micropogonias furnieri)]. Group 2 comprised of sixty (60) farm raised fish [40

marine fish, sea bream (Sparus aurata), 20 freshwater fish; 10 Nile tilapia (Oreochromis

niloticus), 10 hybrid red tilapia (Oreochromis spp.)]. Group 3 comprised of ten (10) caught fish

[grey mullet (Mugil cephalus)].

Frozen fish were originated from 3 countries, namely, Argentine, Uruguay and Vietnam and

the labele information indicating the country of origin, belly gutted or non-gutted fish, date of

production, date of expiry, weight and storage temperature (-180 oC). Farm-raised fish were

collected directly from four commercial fish farms A, B, C and D, located in Gaza, Middle and

Khan Yunis governorates. Sampling was done from November 2016 to March 2017. The sea

bream and the two tilapia species had been cultivated in land based ponds and feed on pellets.

Mugil cephalus were caught by local fishermen from the shoreline of Gaza city, then

transported to the market where they were presented for customers.

3.4 Fish transport and handling

Frozen fish, farmed fish and wild caught fish were randomly purchased from supermarkets, fish

farms and fish markets respectively. Each fish was placed individually in sterile bags and

labeled with the necessary data (date, time of collection, fish type, source, etc...,) Fish samples

were transported immediately or within 2 hours after collection from sampling locations in

cooler boxes filled with ice to Public Health Laboratory-Palestinian Ministry of Health, where

they immediately analyzed.

Upon arrival, the fish were examined under aseptic conditions. Frozen fish were thawed at room

temperature for one hour. The biometric data such as the standard lengths (cm) and the body-

37

wet weights (g) of each fish were measured. After biometric data recording, fish were prepared

for antibiotic residual analysis and microbiological quality testing. Muscle samples were taken

with sterile tools from the dorsal part of fish. While the interior flesh was obtained for antibiotic

residual detection, the muscles with the skin (edible parts) were aseptically collected for

microbiological quality examination.

3.5 Antibiotic residues examination

Fish samples were tested for the occurrence of antibiotic residues by microbial inhibition

technique.

3.5.1 Principle of the test

The test depends on preparing plates seeded with sensitive strain of bacteria at certain

circumstances. The presence of antimicrobial residues in the sample will prevent the growth

of the bacterial organism as indicated by the presence of inhibition areas on the seeded plates.

3.5.2 Buffer preparation

Four kinds of potassium phosphate buffers were prepared using dipotassium hydrogen

phosphate (K2HPO4) and Potassium dihydrogen phosphate (KH2PO4) (USDA, 2013) as shown

in (Table 3.4).

Table (3.4): Preparation of Phosphate Buffers with different pH values

Buffer strength K2HPO4 (gm./L) KH2PO4 (gm./L)

(A) 0.1M Phosphate buffer solution pH 4.5 ------------ 13.61

(B) 0.1M Phosphate buffer solution pH 6.0 2.8 11.2

(C) 0.1M Phosphate buffer solution pH 8.0 16.73 0.523

(D) 0.2M Phosphate buffer solution pH 8.0 33.46 1.046

For each chemical, the required weight was dissolved in 800 ml of distilled water. The pH of

the solution was adjusted if required by the drop wise addition of 0.1 N HCl or 0.1 N NaOH.

Using a volumetric flask, solutions were diluted up to 1 liter. Buffers were autoclaved for 15

minutes at 121°C.

38

3.5.3 Sample preparation and storage

Fish were handled so that freezing and thawing were kept to a minimum. For each fish, four

sterile bags; each with a different pH buffer, pH 4.5, 0.1M; pH 6, 0.1M; pH 8, 0.2M and pH 8,

0.1M were used to identify tetracyclines, β-lactams, macrolides and aminoglycosides residues

respectively. The bags were labeled with the sample and buffer pH information.

Five grams amount of fish muscle sample were weighed and put into a sterile bag, smashed

thoroughly by a stomacher (AES, laboratoire, France) to create a homogenous paste, then

20±0.5 ml of the appropriate buffer was added into the bag. After homogenization, the samples

were allowed to settle for a minimum of 45 minutes before use. Supernatant fluid was collected

into a set of Eppendorf tubes. The fish extracts were refrigerated if they were kept for more

than 2 hours before use. The extracts may be stored in the refrigerator for 24 hours, or frozen

for 14 days for more testing (USDA, 2011).

3.5.4 Preparation of bacterial suspensions:

KWIK-STIK™ is a self-contained package containing a lyophilized microorganism pellet,

reservoir of hydrating fluid, and inoculating swab (Figure 3.2). Bacteria were cultured

according to manufacturer's instructions (Microbiologics). Briefly, the ampoule at the top of

the KWIK-STIK was pinched to release the hydrating fluid which will flow through shaft into

the bottom of the stick hydrating the microorganism pellet. The pellet was crushed by hand to

mix the inoculum and to obtain homogenate suspension. The cap of the device was opened and

the swab with the hydrated material was gently removed. The heavily saturated swab was used

to transfer the hydrated material to the agar. The agar was inoculated by gently rolling the swab

onto one-third of Muller Hinton agar plates. A sterile loop was used to streak the plate to obtain

isolated colonies. Plates were incubated at 37°C for 24 hours.

After the incubation period, a well isolated colony was picked by a sterile loop and streaked

onto a nutrient agar slant then incubated at 37 °C for 24 hours. A sterile nutrient broth was

aseptically added to slants, which tilted softly to free colonies from agar surface. Then, the

suspension was collected into a nutrient broth-containing tube and standardized to have an

absorbance of 0.36 at 600 nm wavelength (Microbiologics, 2017).

39

Figure (3.1): Lyophilized bacterial strains that was used in Inhibition assay (Microbiologics,

2017)

3.5.5 Preparation of Bioassay Plates

Bioassay plates were prepared according to Food Safety Inspection Services (FSIS) protocol

(USDA, 2011) with slight modification. Three antibiotic assay media (4, 8 and 11) were

prepared according to manufacturer's instructions (HiMedia) and autoclaved at 15 psi, 121°C

for 15 minutes. After autoclaving, media bottles were cooled in water bath pre-adjusted at 48°C.

A specific volume of bacterial suspension was inoculated into tempered, melted antibiotic assay

media to produce the desired concentration of bacteria that form a confluent growth on a Petri

plate (Table 3.5).

Table (3.5): Bacterial suspension concentrations in plates

Plate

number

Microorganism Suspension

/100ml

Medium

1 B. cereus 300 µl 8

2 K. rhizophila 680 µl 4

3 K. rhizophila 680 µl 4*

4 K. rhizophila 680 µl 11

5 S. epidermidis 250 µl 11

*without penicillinase

The bottles were thoroughly mixed by swirling and then using a 20 ml syringe, 6 ml of the

seeded media were poured into sterile Petri dishes (diameter 90 mm) to form a layer of 1 mm

thickness. Plates were gently rotated to ensure a uniform distribution of the medium on the

surface of the plate and then kept at 2 – 8ºC, where they were used within 5 days of preparation

(USDA, 2011).

61

Five different inoculation plates each with different medium were used for antibiotic residue

detection. Except for plate number 3, which has no penicillinase, a volume of 1 ±0.1 ml of

penicillinase (BD) enzyme per 100 ml of seeded medium (100,000 units per ml of agar) was

added to all other plates. Plates # 1 (medium 8) have a test agar pH of 4.5 and seeded with B.

cereus; plates # 2 (medium 4), test agar pH 6, seeded with K. rhizophila; plates # 3 (medium

4), test agar pH 6, seeded with K. rhizophila but without penicillinase; plates # 4 (medium 11),

test agar pH 8, seeded with K. rhizophila ; and plates #5 (medium 11), test agar pH 8, seeded

with S. epidermidis (Table 3.5 and Table 3.6).

3.5.6 Assay procedures:

Standard stainless steel bioassay cylinders were used to apply the fish tissue extract on agar

surface; these cylinders are 10 mm high, 6 mm inside diameter and 8 mm outside diameter as

shown in (Figure 3.1).

Figure (3.2): Standard stainless steel bioassay cylinders

Five stainless steel cylinders were placed on the surface of each plate as shown in Figure (3.3).

A 200 µl of fish extract was added into the cylinders by 200 µl micropipette. Each extract was

added into bioassay cylinders on the five prepared petri plates as indicated in Table (3.6). In

addition to sample extracts, an antibiotic sensitivity disk for each antibiotic group was added

on the certain plate for that group.

Figure (3.3): Stainless steel bioassay cylinders showing variation in zones of inhibitions

62

Table (3.6): The pH and Antibiotic disks according to plate number assigned for the

Bioassay

Plate number pH of extraction buffer Antibiotic disk Symbol and potency

1 4.5 0.1 M Tetracycline Te (30)

2 6 0.1M Penicillin P (10)

3* 6 0.1M Penicillin P (10)

4 8 0.2M Erythromycin E (15)

5 8 0.1M Neomycin N (5)

* Without penicillinase.

Plates from 1 to 4 were incubated at 29 ±1ºC for 16 to 18 h., while plate number 5 was incubated

at 37 ±1ºC for 16 to 18 h. After incubation, the occurrence or absence of zones of inhibition on

every plate was observed and recorded (USDA, 2011).

3.5.7 Results interpretation

Results depends on four basic factors; the kind of microorganism, type of media, presence or

absence of penicillinase, and the levels of antimicrobial residue. These factors are listed in

(Table 3.7) (USDA, 2011).

3.5.7.1 Identification of tetracyclines residues

The tetracycline's can be tentatively identified by zones of inhibition (ZI) > 8 mm on Plate 1.

When the concentration of tetracycline is low, there may be no ZI on any other plate. Higher

concentrations of tetracyclines may create zones on all the plates except 5.

3.5.7.2 Identification of β-Lactams residues

The occurrence of β-lactams antibiotics such as penicillin in a sample is identified by ZI ≥ 8

mm on plate 2 and no zone on other plates.

3.5.7.3 Identification of Macrolides residues

The presence of macrolides such as erythromycin and tylosin in tissue is indicated out by ZI >

8 mm on plate 4. When the concentration of erythromycin is high, inhibition zones may

appear on all other plates.

61

3.5.7.4 Identification of Aminoglycosides residues

Neomycin and gentamicin residues occur in tissue at low concentrations (8 mm only on Plate

5. At higher concentrations, neomycin and gentamicin may also form zones of inhibition on

plates 1 and 4 but not on 2 and 3.

Table (3.7): Interpretation of results of five-plate bioassay (USDA, 2011)

Pla

te No

an

d

Micro

org

an

ism

Ag

ar

Tetra

cyclin

e's

β-la

ctam

s

Ma

crolid

es

Am

inog

lyco

sides

H L H & L H L H L

1- B. Cereus ATCC 11778 8 S S R S R S R

2-K. rhizophila ATCC 9341a 4* S R S S R R R

3-K. rhizophila ATCC 9341a 4 S R R S R R R

4-K. rhizophila ATCC 9341a 11 S R R S S S R

5- S. epidermidis ATCC

12228

11 R R R S S S S

H = High concentration

L = Low concentration S = Zone of inhibition R = No zone of inhibition

*= without penicillinase

3.6 Microbial analysis

During the study, total viable count, S. aureus and total coliforms and presence of pathogenic

organisms (namely, Salmonella spp. and Vibrio spp.) of public health significance from the

fishes were investigated.

3.6.1 Sample preparation

Ten-gram samples of the edible part of the fish, that is the flesh or muscle with skin was

aseptically cut from dorsal side of each fish with a sterile knife and weighed in sterile petri dish.

Each sample was homogenized with 90 ml of sterile phosphate buffer solution using a

stomacher (AES, laboratoire, France). Tenfold dilutions of the homogenates up to 10-5 were

made in normal saline using automatic pipette (Kumar et al., 2014).

3.6.2 Total viable count (TVC)

The total bacterial count of the fish samples was determined by following the conventional pour

plate method (Anon, 1992). Ten serial dilutions of fish samples were prepared in test tubes then

63

1 ml of each dilution was transferred into sterile glass petri dishes. Approximately 10 ml of

melted nutrient agar medium (45-50°C) was poured into each plate, where they thoroughly

mixed and left 10 min for solidification. The plates were incubated at 30°C for 48 hours. After

the incubation period, developed colonies were counted per each plate of the same dilution. The

total colonies count per gram of samples was calculated as follows:

Total viable bacterial count = average number of triplicate plates of the same dilution x

reciprocal of the dilution used colony forming unit (CFU)/g sample.

This was done in triplicates for each fish sample and the average was taken.

3.6.3 Total coliform count

Decimal dilutions (10-2, 10-3, and 10-4) of fish extract (prepared as described in section 3.6.1)

were used. One ml of each dilution was transferred aseptically into separate, Petri dishes in

duplicate. 12-15 ml of melted (and cooled to 45°C) Violet Red Bile Agar (VRBA) were added

to each plate. The plates were swirled gently and after the agar medium solidified, the plates

were incubated for 18 to 24 h at 37°C. To prevent surface growth and spreading of colonies,

the plates were overlaid with 5 ml VRBA, and let to solidify. Pink colonies with diameter 2-3

mm after incubation period were counted (Bej et al., 1990); (Kumar et al., 2014).

3.6.4 Staphylococcus aureus

Two decimal dilutions (10-2, 10-3, and 10-4) of fish extracts were used. One ml of each dilution

was aseptically transferred and spread on the surface of Baird Parker agar using sterile L-shaped

glass rod. Plates were incubated for 45-48 h at 35 ºC. Typical Staphylococcus aureus colonies

will appear circular, smooth, convex, moist, 2-3 mm in diameter on uncrowned plates, gray to

jet-black, frequently with light colored margin, surrounded by opaque zone and frequently with

an outer clear zone. S. aureus is coagulase and catalase positive. Suspected colonies were

stained with Gram staining. S. aureus is Gram positive cocci. (FDA, 1998).

3.6.5 Detection of Salmonella

Nonselective pre-enrichment:

Salmonella was isolated following a published protocol with some modifications (Bakr,

Hazzah, & Abaza, 2011). Briefly, a 25 g of each fish sample was homogenized with 225 ml of

buffered peptone broth using a stomacher (AES, laboratoire, France) for one minute. Diluted

samples were incubated for 18- 20 h at 35 ºC to provide suitable conditions required for the

survival and repair of stressed and injured Salmonella cells.

66

Selective enrichment: About 0.1 mL of the nonselective pre-enriched sample was transferred

to a tube containing 10 ml of Rappaport-Vassiliadis broth (RV). Similarly, 1 ml of the pre-

enriched sample was transferred to 10 ml of tetrathionate broth (TT). Both media were

incubated at 42 ºC for 24 hours.

Plating on solid selective media: Tubes of selective enrichment media were shaken and then

a loopful from each was streaked onto the surface of CHROMagar Salmonella Plus medium

and Xylose lysine desoxycholate (XLD) agar. Plates were incubated at 35 °C for 24 hours and

then examined for typical Salmonella colonies.

Identification of Salmonella spp.: The identification of suspected Salmonella colonies (pink

or reddish color with black center on XLD) was done morphologically and biochemically using

API20 system (Biomerieux, France). For serological identification, isolates inoculated on

nutrient agar slants and incubated at 37 °C for 24 hours. Pure colonies were emulsified in one

drop of sterile normal saline, on a glass slide and a drop of polyvalent Salmonella O -antisera

was added, and observed for agglutination .

3.6.6 Detection of Vibrio spp.

Enrichment: Twenty-five grams of each fish sample was weighed and mixed with a small

amount of alkaline peptone water (APW) in a sterile stomacher bag. The bag was blended

thoroughly. After blending, APW was be added to make the total amount of APW equal to 225

ml (10-1 dilution). Two dilutions (10-2 and 10-3) of the blended samples were prepared in APW

and incubated at 35˚C for 6 to 8 hours.

Culture on a selective medium: were inoculated using a loopful from the enrichment culture

was streaked onto thiosulphate Citrate Bile Salt Sucrose (TCBS) agar plates and incubated at

35˚C for 18 to 24 hours.

Identification of Vibrio spp: Suspected Vibrio spp. colonies from TCBS plates (yellow or

green colonies) were examined according to conventional biochemical characteristics

including: oxidase test, subculture to peptone water with sodium chloride (NaCl) of different

concentrations (0-3-6-8-10%), arginine dehydrolase, lysine and ornithine decarboxylase tests,

growth at 42˚C, Voges proskauer test, urease test, and gelatinase test (Bakr et al., 2011).

65

3.7 Questionnaire

A close-ended questionnaire was used to collect data about using antimicrobials in fish farming.

Questions included if fish farmers have knowledge about instructions of the used drugs and the

withdrawal period of these drugs. Questionnaires were completed through interview with

farmers or owners. See (Annex 1 and 2) for Arabic and English version of the questionnaire.

3.8 Data analysis

Data generated from the analysis of fish samples and from the questionnaire survey were

entered into an Excel spreadsheet and then uploaded to SPSS software, version 22 (SPSS Inc.,

USA) for analysis. Collected data were analyzed using descriptive statistics as appropriate,

including: frequency, means, ranges, proportions and standard deviations. Data were also cross

tabulated and the Chi square test was used to detect significant differences among various

variables. P values under 0.05 were considered significant.

64

Chapter IV

Results

67

Chapter IV

Results

This chapter presents the main findings of the study. It is divided into three major sections. The

first section deals with biometric measurements, the second deals the presence of antibiotic

residues in fish samples while the third presents the findings of microbial quality of the tested

fish.

One hundred fish samples (frozen, fresh, and farmed) were collected and analyzed during the

period from Nov. 2016 to May 2017. Argentine hake, croaker fish, sutchi catfish fillet, grey

mullet, sea bream, red tilapia and Nile tilapia fish species were selected because they are the

most commonly consumed fish in Gaza Strip.

The investigation included the detection of the residues of four antibiotic groups; β-Lactams,

macrolides, aminoglycosides and tetracycline's. In addition, examination of microbiological

indicators including; Total Plate Count (TPC), Total Coliform (TC), Staphylococcus aureus

count and the occurrence of Salmonella spp., and Vibrio spp. was performed.

4.1 Biometric measurements

The biometric characteristics, including length (measured as total length) and weight of fish

recorded after the collection of fishes are listed in Table 4.1 with their common name and the

type to which they belong. The mean body weight of investigated fishes was ranged from 145.7

to 950.7g, and the mean total length was ranged from 20.0 to 45.0cm

Table (4.1) List of fish species collected from local markets and farms with means, standard

deviations and ranges of weight (g) and length (cm).

Total length(cm) Weight(g)

No. Common name Fish type Range

(min. – max.) Mean ± SD

Range

(min. – max.) Mean ± SD

ND ND ND ND 10 Argentine hake

Imported frozen 35.2-45.0 39.1± 3.1 568.0-950.7 716.0±120.0 10 Croaker fish

ND ND ND ND 10 Sutchi catfish fillet

25.0-30.0 27.8±1.7 145.7-206.6 176.1±21.6 10 Grey mullet Wiled caught

24.0-29.0 26.8±1.3 215.9-398.0 355.0±32.1 40 Seabream

Farmed 20.0-26.0 11.1 ±1.9 242.0- 271.2 .124 1 ± 33.7 10 Red tilapia

20.0-24.0 11.2 ± 1.5 286.5 – 240.0 .127 6 ± 18.1 10 Nile tilapia

ND, not determined

68

4.2 Detection of antibiotic residues

Fish samples were examined for the residues of four antibiotic groups namely β-Lactams,

macrolides, aminoglycosides and tetracyclines. The sample was considered positive (containing

residues) when its extract inhibits the growth of bacteria on any plate with a zone of inhibition

more than 8 mm in diameter. Table 4.2 shows the result of analysis of antibiotic residues in all

investigated fish with prioritization of antimicrobials categorized by World Health

Organization as critically important and highly important (WHO, 2017). Residue screening

tests indicated that β-lactams and macrolides were not detected in any of the examined fish. On

the other hand, a total of 53% (53/100) of fish were found to contain antibiotic residues, with

aminoglycosides were the most frequently observed followed by tetracyclines. There were 52

(52%) of fish detected as being contaminated with aminoglycosides residues and only one fish

1(1%) sample (the frozen Sutchi catfish fillet) that showed positive results for the presence of

tetracyclines residues (Table 4.2).

Table (4.2) Antibiotics detected in different fishes with prioritization of antimicrobials

categorized as Critically Important and Highly Important.

Class Representative

Antibiotic

Number (%) of fish in

which antibiotic is

detected

On the WHO’s critically

important antimicrobials list

(2016)

β-Lactams Penicillin 0 (0.0%) Critically important

Macrolides Erythromycin 0 (0.0%) Critically important

Aminoglycosides Neomycin 52 (52.0%) Critically important

Tetracyclines Tetracycline 1(1.0%) Highly important

4.2.1 Aminoglycosides residues

A Comparison of percentages of different types of fish (locally farmed, frozen imported and

wild caught fish) which were found to contain aminoglycosides residues is presented in Figure

(4.2). Of the 60 farmed fish analyzed in this study, 52 (86.7%) were found to contain detectable

levels of aminoglycosides residues. At the same time, all (n = 30) imported frozen fish

(Argentine hake, croaker fish and sutchi catfish fillet), and the ten wild caught fish (grey mullet)

were negative for aminoglycosides .

69

Figure (4.1): Comparison of percentages of farmed, frozen imported and wild caught fish

which were found to contain aminoglycosides residues.

Among the locally farmed fishes 87.5% (35/40) of sea bream, 70% (7/10) of Nile tilapia and

all (100%, 10/10) of red tilapia were positive for aminoglycoside (Table 4.3). While none of

the sea bream from farm A was found to be negative for aminoglycoside residues, negative

results were found in fish from farms B (one fish, 7.19%) and C (four fish, 30.8%).

Table (4.3) Aminoglycoside residues screening results of analyzed Fish

Aminoglycoside screening results

Common name Positive Negative

% No. % No.

0% 0 100% 10 Argentine hake

0% 0 100% 10 Croaker fish

0 0 100 10 Sutchi catfish fillet

0% 0 100% 10 Grey mullet

100 13 0 0 Seabream (Farm A)

92.9% 13 7.19% 1 Seabream (Farm B)

69.2% 9 30.8% 4 Seabream (Farm C)

70 7 30 3 Nile tilapia (Farm D)

100 10 0 0 Red tilapia (Farm D)

52% 52 48% 48 Total

4.3 Microbiological indicators

The microbial indicators including, total plate counts, total coliform count and S. aureus count

are presented in Table 4.4 as colony forming unit (cfu/g) for the different fish species and fish

groups (frozen, wild caught and farmed fish). The microbiological quality of fish varied widely

between the different fish groups. The microbial load was highest in locally farmed fish species,

86.67

0.00 0.000.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

Farmed Frozen imported Wild caught

Pe

rce

nta

ge (

%)

of

po

siti

ve

amin

ogl

yco

sid

es

resi

du

es

Fish group

51

followed by the wild caught fish species (grey mullet) and then the imported frozen fish. Among

farmed fish species, the microbial load of the freshwater fish species, Nile tilapia was the

highest (TPC, 2×105-1×106 cfu/g) followed by the congeneric species red tilapia (TPC, 4×104-

5×105 cfu/g) and then the marine fish species seabream (TPC, 0-5×105 cfu/g). The number of

total coliform in red tilapia ranged from 2×104 to 1×105 cfu/g, counts higher than those found

in seabream, grey mullet and the imported, frozen fish species, sutchi catfish fillet where the

counts were ranged; 2×103-4×104, 0-1×105 and 0-1.3×103 cfu/g respectively. S. aureus was

detected in Nile and red tilapia only.

Table (4.4): Ranges of total plate count, total coliform count and S. aureus count expressed as

colony forming unit (cfu/g) in frozen, wild caught and farmed fish species.

Fish Type Fish Name (n) TPC (cfu/g)

Total coliform

(cfu/g)

S. aureus

(cfu/g)

Frozen Argentine hake (10) 0-5×102 0 0

Croaker fish (10) 1.0×102-5×103 0 0

Sutchi catfish fillet (10) 3×102-15×102 0-4×102 0

Wild caught fish Grey mullet (10) 35×102-50×103 0-1.3×103 0

Farmed fish

Seabream (Farm A)(13) 0-2.5×104 0 0

Seabream (Farm B)(14) 0-2×104 0-4×103 0

Seabream (Farm C)(13) 0-5×105 0-1×105 0

Nile tilapia (Farm D)(10) 2×105-1×106 2×103-4×104 0-4×105

Red tilapia (Farm D)(10) 4×104-5×105 2×104-1×105 0-6×103

A summary of the number of positively and negatively contaminated fish, the prevalence of

bacterial contamination and the occurrence of simultaneous multiple contamination

(contamination to more than one bacterial indicator) in the different types of fish is presented

in Table 4.5

Investigated fish revealed a variable contamination pattern. Out of all 100 fish included in the

study, 85% were positive for bacterial contamination, with prevalence of 73.3, 100 and 88.3%

in frozen, wild caught and farmed fish respectively. Among the one hundred tested fish, 44, 25

and 16% showed single, double and triple contamination with the tested bacterial indicators

respectively

52

Table (4.5): Number of positively and negatively contaminated fish, the prevalence of bacterial

contamination and the occurrence of multiple contamination in the different types of fish

Fish Type No. of

fish

Occurrence of

contamination (no.)

Prevalence

(%)

Occurrence of simultaneous

multiple contamination

negative positive Single Double Triple

Frozen fish 30 8 22 73.3 21 1 0

Wild caught fish 10 0 10 100 8 2 0

Farmed fish 60 7 53 88.3 15 22 16

Total 100 15 85 85 44 25 16

For frozen fish, 21 (70%) and 1 (3.3%) were singly and doubly contaminated respectively. Similarly, 8

(80%) and 2 (20%) of the wild caught fish were singly and doubly contaminated respectively. Farmed

fish showed single, double and triple contamination of 25, 36.7 and 26.7% respectively (Table 4.5 &

Figure 4.2).

Figure (4.2): Number and percentage of negativly and positevely (single, double and triple

contamination) contaminated fish based on their type.

All (100%) croaker fish, Sutchi catfish fillet, Grey mullet, Nile and red tilapia fish as well as,

82.5% of sea bream, and 20% of Argentine hake were positively contaminated. Nile and red tilapia

were the most contaminated species with 10 ; 40 and, 90; 60% double and triple microbial

contaminations respectively (Table 4.6).

51

Table (4.6): Number and percentage of negativly and positevely (single, double and triple

contamination) contaminated fish species.

Occurrence of contamination

Fish Name Fish Type

Positive Negative

Multiple contamination

(%)

% No. % No.

Triple double single

0 0 20 20 2 80 8 Argentine hake

Frozen 0 0 100 100 10 0 0 croaker fish

0 90 10 100 10 0 0 Sutchi catfish fillet

0 20 80 100 10 0 0 Grey mullet Wild caught fish

2.5 42.5 37.5 82.5 33 17.5 7 Seabream

Farmed fish 90 10 0 100 10 0 0 Nile tilapia

60 40 0 100 10 0 0 Red tilapia

85 15 Total

The compliance with Palestinian fish safety criteria for the five microbial indicators collectively

is presented in Table 4.7. Despite the high prevalence of contamination among investigated fish

(85%), only 39% failed the standard at some points, with the majority of failed fish belonged

to farmed fish. All Nile and red tilapia and 42.5% of the Sea bream fish failed to comply with

the standards. On the other hand, only10% of both, the wild caught fish species and sutchi

catfish fillet were failed the standards.

Table (4.7): The compliance with the Palestinian microbiological standard for fish.

Compliance with Palestinian standards

Fish Name

Fish Type Failed Passed

% N % N

0 0 100 10 Argentine hake

Frozen 0 0 100 10 Croaker fish

10 1 90 9 Sutchi catfish fillet

10 1 90 9 Grey mullet Wild caught fish

42.5 17 57.5 23 Seabream

Farmed fish 100 10 0 0 Nile tilapia

100 10 0 0 Red tilapia

39 39 61 61 Total

4.3.1 Total plate count

The compliance with the Palestinian stands for total plate count is shown in Figure (4.3).

Ninety six percent of investigated fish were within the acceptable range of Total plate count

standard specification. On the other hand, 4% were unacceptable as they yielded over 106

cfu/g.

53

Figure (4.3): Percentage of fish that complied with the Palestinian Standards for TPC.

Table (4.4) shows the compliance with total plate count (TPC) standards of frozen, wild caught

and farmed fish species. Three of Nile tilapia (30%), and one (10%) of red tilapia exceeded the

standard specification while the rest of fish samples were within the standard specification.

Table (4.8): The compliance with total plate count (TPC) standards of frozen, wild caught

and farmed fish species.

Compliance wit h TPC

standards

Fish Name

Fish Type

Failed Passed

% N % N

0 0 0 10 Argentine hake

Frozen 0 0 100 10 Croaker fish

0 0 100 10 Sutchi catfish fillet

0 0 100 10 Grey mullet Wild caught fish

0 0 100 13 Seabream (Farm A)

Farmed fish

0 0 100 14 Seabream (Farm B)

0 0 100 13 Seabream (Farm C)

30 3 70 7 Nile tilapia (Farm D)

10 1 90 9 Red tilapia (Farm D)

4 4 96 96 Total

Figure (4.4) shows the number and percentage of the different types of fish that pass and fail

the Palestinian Standards for TPC. Four (33.3%) of farmed fish exceeded the standard

specification while all the rest were within the limits. The chi square test indicated a significant

difference in the total number of positive samples for TPC among the different fish types (p

value = 0.004).

96 (96%)

4 (4%)

Pass Failed

Nu

mb

er

of

fish

compliance with palestinian standard

56

Figure (4.4) Number and percentage of the different types of fish that pass and fail the

Palestinian Standards for TPC.

4.3.2 Total coliform

As shown in Figure (4.5), 61 out of the hundred fish examined for total coliform bacteria were

complied with the Palestinian standard, while 39% were above the standard limits.

Figure (4.5): Percentage of fish sample that compiled the Palestinian standard for total

coliform.

Table (4.9) presents the compliance with total coliform count standards of frozen, wild caught

and farmed fish species. It can be noted that, only one (10%) of sutchi catfish fillet and one

(10%) of grey mullet, 17 (42.5%) of seabream (from Farms B and C), 10 (100%) of Nile tilapia

and 10 (100%) of red tilapia were higher than the limits recommended by the Palestinian

standards.

55

Table (4.9): The compliance with total coliform standards of frozen, wild caught and farmed

fish species.

Complied with coliform

standards Fish name Fish type

Failed Passed

% N % N

0 0 100 10 Argentine hake

Frozen fish 0 0 100 10 Croaker fish

10 1 90 9 Sutchi catfish fillet

10 1 90 9 Grey mullet Wild caught fish

0 0 100 13 Seabream (Farm A)

Farmed fish

71.4 10 28.6 4 Seabream (Farm B)

53.8 7 46.2 6 Seabream (Farm C)

42.5 17 57.5 23 Seabream (total)

100 10 0 0 Nile tilapia (Farm D)

100 10 0 0 Red tilapia (Farm D)

39 39 61 61 Total

Figure (4.6) shows that one (3.3%) frozen fish, one (10%) wild caught fish and 37(61.7%) of

farmed fish were above the standard specification. There was a significant difference in the

coliform count between imported frozen, wild caught and locally farmed fish (chi square test,

p < 0 .001).

Figure (4.6): Number and percentage of the different types of fish that pass and fail the

Palestinian Standards for total coliform.

54

4.3.3 Staphylococcus aureus

Results reveals that 87% of samples were within the acceptable range of recommended limits

of S. aureus and only 13% of examined fish were above the limits (Figure 4.6).

Figure (4.7): Percentage of fish that complied with the Palestinian Standards for S. aureus.

Table (4.10) shows the compliance with S. aureus standards for the three fish types; frozen,

wild caught and farmed fish species. Eight Nile tilapia and five of red tilapia exceeded the

standard specification while the remaining were within the standard limits.

Table (4.10): The compliance with S. aureus standards of frozen, wild caught and farmed fish

species.

Compliance with S. aureus

standards Fish name Fish Type

Failed Passed

% N % N

0 0 100 10 Argentine hake

Frozen fish 0 0 100 10 Croaker fish

0 0 100 10 Sutchi catfish fillet

0 0 100 10 Grey mullet Wild caught fish

0 0 100 13 Seabream (source A)

Farmed fish

0 0 100 14 Seabream (source B)

0 0 100 13 Seabream (source C)

80 8 20 2 Nile tilapia (source D)

50 5 50 5 Red tilapia (source D)

13 13 87 87 Total

57

Results presented in Figure (4.8) show that 13 (21.7%) of the farmed fish exceeded the standard

specification. At the same time 78.3% of farmed fish, and all (100%) of frozen and wild caught

fish were within the standard specification for S. aureus. According to chi square test, there was

a significant difference between the levels of S. aureus of the different fish type (p value =

.007).

Figure (4.8): Number and percentage of the different types of fish that pass and fail the

Palestinian Standards for S. aureus.

4.3.4 Salmonella

Out of one hundred fish tested for Salmonella, only one sample of seabream (Farm C) showed

a positive result while the rest were negative.

4.3.5 Vibrio spp.

All tested fish were negative for Vibrio spp.

4.4 Questionnaire results

Four farms (A, B, C, D) were chosen and registered in the study. One farmer (Farm C) refused

to participate. Among the selected farms, three raised sea bream and one farm raised Nile

tilapia and red tilapia. The questionnaire is concerned essentially about antimicrobials usage in

fish farms.

58

4.4.1 Use of Antibiotics in Surveyed Farms

One farmer reported the he use antibiotics during cultivating period of fish and the rest farms

reported not to use antibiotics during the surveyed period the main farmer's responses are

presented in (Table 4.11).

Table (4.11): Farmer's responses to the questionnaire.

Subject Yes % No %

I use antimicrobials on fish for therapy. 1 33.3 2 66.6

I use antimicrobials on fish for prevention. 0 0 3 100

I have good knowledge of the nature of all used drugs. 1 33.3 2 66.6

I have good knowledge of the instructions of the used drugs. 1 33.3 2 66.6

I have good knowledge of the WP of used drugs. 1 33.3 2 66.6

I accelerate healing by using a double dose of drugs. 0 0 3 100

I use more than one antimicrobial in a single treatment. 0 0 3 100

I am aware of the negative effect of drug residues on human health. 2 66.6 1 33.3

I do sell fish during a treatment period. 0 0 3 100

I do have a license for the farm 3 100 0 0

Farmers were asked about consulting veterinarians, method of antibiotic administration and

time of marketing after treatment. Farmer's responses are shown in (Table 4.12).

Table: (4.12): farmer's behaviors in dealing with antibiotics in farms

Subject Options Response of farmer's Percentage

the reason for using

antimicrobials

Prevention 1 33.3

Treatment 2 66.6

Enhancement 0 0

Consulting

veterinarians

Always 0 0

Sometime 0 0

Never 3 100

Method of antibiotic

administration

Pond water 0 0

Feed 1 33.3

Stop giving drugs

before marketing.

One day 0 0

Two days 0 0

More 1 33.3

water pond renewal Continuous renewal 2 66.6

Every day 1 33.3

Every week 0 0

59

Chapter V

Discussion

41

Chapter V

Discussion

In this study the antibiotic residues and microbial quality of fish were examined. To the best of

our knowledge, this is the first study undertaken to investigate the presence of antibiotic

residues and the microbiological quality of some fish presented for direct human consumption

in the local markets of the Gaza strip and to determine whether fish products consumed in Gaza

strip is safe for human consumption. Argentine hake, Croaker fish, Sutchi catfish fillet, Grey

mullet, Sea bream, Red tilapia and Nile tilapia fish species were selected because they represent

some of the most commonly consumed fish in Gaza Strip.

5.1 Antibiotic residues

The existence of antibiotic residues in foods of animal origin such as meat and fish has attracted

attention from local, national and international public health agencies (Ašperger et al., 2009).

Several studies have reported that the antibiotic resistance acquired by large number of bacterial

species may be due to exposure to these drugs and resistance genes may be transmitted to human

and/or fish pathogens (Grigorakis & Rigos, 2011). In addition, human consumption of a large

quantity of fish and fish products with antibiotic residues may cause unfavorable changes in

intestinal microbiota and create immunological response reactions in susceptible persons

(Mottier, Parisod, Gremaud, Guy, & Stadler, 2003).

The results of the present study are of high concern, since they revealed the presence of

antibiotic residues in most (86.7%) of the domestically farmed fish species such as gilthead sea

bream red and Nile tilapia sold in Gaza strip, which may form a potential risk to fish consumers.

Although antibiotic residues found at the farm level, but these fish were ready for sale and

marketing.

Aminoglycosides and tetracycline's detected in the screened fish are very important to human

health and medicine. Due to this importance, they are still found on the list of the 2016 revised

World Health Organization (WHO) of critically important antimicrobials for human medicine

(WHO CIA list) where they classified as critically important and highly important

antimicrobials respectively (Table 4.2). “The list is intended to assist in managing antimicrobial

resistance, ensuring that all antimicrobials ,especially critically important antimicrobials, are

used prudently both in human and veterinary medicine” (WHO, 2017).

42

Antibiotic residues were detected in 53/100 (53%) of the screened fish. The present study used

the microbiological inhibition tests for screening of antibiotic residues in fish, so it was difficult

to predict whether the detected levels were beyond the maximum residue limits (MRLs) or not.

In fact, the validation of a microbiological based method for the screening of antibiotic residues

is still controversial. For instance, following a microbiological inhibition test for antibiotic

residues in spiked milk samples, (Gaudin et al., 2004) found that, out of the 66 screened

antibiotics, 48 (75%) were detected below the MRL up to four times the MRLs, while the

remaining were either detected between four and 150MRLs or had no MRLs at all. In order to

exactly identify and quantify the antimicrobial residues in a certain type of biological sample

however, a confirmatory methods like the liquid chromatography or gas chromatography are

essential (Balizs & Hewitt, 2003). These methods were not used in this study due to its

complexity and expensive cost. Considering the cheapness, the simplicity to perform and the

broad spectrum characteristics, the microbiological-based methods is still a good option for

detection of a wide range of antibiotics with satisfactory sensitivity (Gaudin et al., 2004). The

three-plate agar diffusion test using Bacillus subtilis BGA as the test bacterium for example

was able to detect penicillin G residues in both kidney and muscle tissues, and that of

enrofloxacin+ciprofloxacin , and oxytetracycline residues in kidney tissue, at about or below

MRL concentrations (Myllyniemi, 2004).

All examined fish were negative for β-lactams and Macrolides. It was only one of frozen fish

(Sutchi catfish fillet), that showed positive results for the presence of tetracyclines residues.

Aminoglycosides, the potentially toxic drugs (Guardabassi & Kruse, 2008), were detected in

52% of all screened fish, which represents 86.7% of the domestically farmed fish group.

It is clear that, fish contamination by antibiotic residues occurred among the farmed fish only,

whether, domestically or abroad. The freshwater sutchi catfish fillet (P. hypothalamus)

available in Gaza markets are originally imported from Vietnam, where they are intensively

raised in cages and pens established in major river tributaries of the Mekong River delta of

Vietnam (Griffith, van Khanh, & Trong, 2010). The inappropriate quality, misuse of antibiotic

and the presence of antibiotic residues in Vietnamese aquaculture products was confirmed by

some previous studies ((Griffith et al., 2010); (Pham et al., 2015)). In European markets, frozen

fish originating from Vietnam has bad reputation in food safety and quality especially with a

medium high microbiological risk level (Noseda, Thi, Rosseel, Devlieghere, & Jacxsens, 2013).

((Noseda et al., 2013); (Pham et al., 2015)) detected the antibiotic residues in one-fourth of

41

tested domestically sold fish in Vietnam, and confirmed the general lack of knowledge about

the purpose and proper usage of antibiotics by aquaculture producers.

The low levels of positive antibiotics residues among the frozen Sutchi catfish fillet (only one

fish) found in this study could be attributed to the denaturation of the residues as a result of the

long term storage of fish at very cold temperatures (Olusola, Folashade, & Ayoade, 2012).

O'brien, Campbell, and Conaghan (1981) however, found various but slight effects (from

minimal to nil) of the cold storage of animal tissues (muscles, kidney and liver) on the biological

activity of the residues of ampicillin, chloramphenicol, oxytetracycline, streptomycin and

sulphadimidine.

In fish farms aminoglycosides are usually used for treatment of Gram-negative and Gram-

positive infections where they selectively inhibit bacterial growth by inhibiting protein

synthesis through binding to the 31s subunit of the ribosome (Sekkin & Kum, 2011).

The presence of antibiotic residues in 86.7% of domestically farmed fish is a clear indication

of the level of misuse of antimicrobial agents by fish producers in Gaza strip. A similar

conclusion concerning the non-prudent use of veterinary drugs by local farmers was obtained

by Elmanama and Albayoumi (2016) who found high prevalence of antibiotic residues among

broiler chickens in the Gaza Strip. The main reason for such antibiotic treatment is mainly to

avoid the morbidity and mortality of farmed animals. It is essential however, that, treated fish

should not be harvested for human consumption until a specified withdrawal time has been

passed.

It seems however, that, the local farmers do not follow the recommended procedures for using

antibiotics at their fish farms and they are not aware regarding the withdrawal times that ensure

there is no detectable antibiotic residue in the fish destined for human consumption. In fish,

withdrawal times are generally depent on the metabolic and excretion rate of drug by a fish.

Because fish are poikilothermic organisms, such rates are in turn depend on the prevailing

environmental conditions, especially the temperature (Haasnoot et al., 1999).

Based on the 500 degree days rule (in which withdrawal period can be calculated by dividing

500 by the mean temperature of the water in degrees Celsius), which was established by

European Economic Commission Directive No. 82/2001 for fish meat (EC, 2001) to represent

the minimum withdrawal period, and the available data of the average daily mean temperature

in Gaza strip, which ranges from 25 oC in summer and 13 oC in winter, it was possible to

43

roughly estimate the required withdrawal time as ≥ 20 days (500/25= 20) in summer and about

42 days (500/13= 41.7) in winter. In fact, these are not so long periods of time to adhere to, if

the ultimate objective is to ensure safety to fish consumers and to protect the health of human

being.

It was suggested however, that the 500 degree days approach is a rough estimate of the

elimination-rate and to be more accurate it was recommended to establish MRLs values for

antibacterial residues in animal products based on pharmacology and the PK of antibacterials

in aquatic species (Sekkin & Kum, 2011).

In recent years, there is increasing evidence that some pharmaceuticals including antibiotics are

present in marine and coastal environments. The major source of antibiotics entering marine

environments are pharmaceutical releases from antibiotic manufacturer, households, and

hospitals via municipal effluent discharges the aquaculture facilities located in the coastal areas

(Gaw, Thomas, & Hutchinson, 2014).

No antibiotic residues were detected in the imported frozen croaker fish (M. furnieri) and the

Argentine hake (M. hubbsi). Perhaps, the reason behind this is that, both are wild marine fish

and may be harvested from antibiotic free fishing ground which makes them less likely exposed

to antibiotic contamination. The marine croaker (M. furnieri) for instance, is distributed from

the Yucatan Peninsula (Gulf of Mexico, 20 N) to the Gulf of San Matias (Argentina, 41 S), and

the geographical distribution of Argentine hake is ranged from Southwestern Atlantic, from

parallel 21°30’S to 49°S to the south and east of the Argentinian coast, and both species

represents an important constituent in the commercial fishing in these areas (Salas,

Chuenpagdee, Charles, & Seijo, 2011).

All locally wild-caught grey mullet (M. cephalus) fish were found negative for the antibiotic

residues, despite the pollution of the seawater as well as the occurrence of high rates of

antibiotic resistance and multiple resistance of clinically important bacteria in the seawater of

Gaza strip was well documented (Elmanama et al., 2016), (Elmanama, Fahd, Afifi, Abdallah,

& Bahr, 2005), (Elmanama, Afifi, & Bahr, 2006), (Afifi, Elmanama, & Shubair, 2000) and

(Elnabris et al., 2013).

46

Fish farming which may constitutes the main source of antibiotic contamination in marine wild

fish (Björklund et al., 1990) is still in its infancy in the Gaza strip to contribute in antibiotic

contamination in locally caught fish.

Moreover, available data indicates that, pharmaceuticals may present in marine and coastal

environments at concentrations that may affect marine organisms at the lower trophic levels

only, such as algae, but there is no evidence for the accumulation of pharmaceuticals in higher

trophic-level organisms of the aquatic food chains such as fish (Gaw et al., 2014).

In conclusion, result of this study shows that some fish farmers in Gaza strip are currently using

antibiotics in their own farms. To avoid the risk associated with using antibiotics however, it is

highly recommended to use other protective measures such as vaccination.

5.2 Microbial quality

Examination of foodborne pathogens in food is playing a significant role in prevention of

foodborne pathogens transmission (WHO, 2010).The microbial quality of fish is an important

aspect of food safety. Raw fish products have been reported as vehicles for foodborne illness

(Uradzński, Wysok, & Gomółka-Pawlicka, 2006). Fishery products are very important for

human nutrition and health benefits worldwide, and can also act as a source of foodborne

pathogens (Kromhout, Bosschieter, & Coulander, 1985) (Darlington & Stone, 2001).

5.2.1 Total plat count

The Palestinian microbiological standards for foods allows TPC, total coliform and S. aureus

count limits for fresh/frozen fish at 106 CFU, 102 CFU and 103 CFU respectively.

In this study, 96% of samples were within the acceptable range of Total plate count standard

specification, while 4% of samples were above the standard of specification. Bacterial load of

all frozen fish were under the acceptable ranges, which indicated good quality of frozen fish,

and good hygiene and clean, during fish processing, throughout all steps, such as catching,

landing, transportation, handling, and preservation. Similar results obtained by Sanjee and

Karim (2016) and Strunjak-Perovic et al. (2010).

45

The bacterial load of the tilapia fish in both species was very high, and it was estimated that it

ranged from 4×104 to 106 Although for the Palestinian species only four samples of black

tilapia and zero for red tilapia were failed, but all samples of black tilapia and six for red tilapia

was failed according to international compliance.

This can be explained by the nature of the life of these fish in fresh water this makes farmers

reduce the number of water ponds renewal times as determined by the owners of the farm in

the questionnaire that was made and this causes the increase of bacterial load on farmed fish.

Higher density of aerobic bacteria was detected in similar study (Adebisi & Emikpe, 2017).

5.2.2 Total coliform

The total coliform test for Argentine hake, Croaker fish and Sea bream (Source A) showed

negative results but Red tilapia have the highest counts ranging from 2 x 104 to 105. The

presence of TC is a possible indication of sewage contamination which may also occur during

different processing steps such as transport and handling. Moreover, the contamination may

also be caused by the water used for washing or icing (Boyd, 1990). The lower number of

coliforms can be due to the effectiveness of safety procedures during processing and handling

(Elhadi, Radu, Chen, & Nishibuchi, 2004).

5.2.3 S. aureus

With regard to S. aureus, Nile tilapia results range from 0 to 4×105 and Red tilapia (source D)

range from 0 to 6000 while the rest of fish types were negative. Similar study conducted by

(Bujjamma & Padmavathi, 2015) to detect the presence of S. aureus in fish collected from local

markets in Guntur, Andhra Pradesh, South India, showed a total of 24.47% of samples

contaminated with S. aureus. In addition, (Saito, Yoshida, Kawano, Shimizu, & Igimi, 2011)

detected S. aureus in fish samples and it was detected in 41 (19.6%) of all tested fish samples.

5.2.4 Salmonella spp.

In this study, only one sample of Sea bream (source C) showed a positive result while the rest

were negative. In contrast, the presence of salmonella was reported in countries like India,

Mexico, Thailand, Hong Kong, Spain and Turkey ((Herrera, Santos, Otero, & García‐López,

2006); (R. Kumar, Surendran, & Thampuran, 2009); (Pamuk et al., 2011)). The highest

presence of Salmonella was detected in fish products was found in Central Pacific and African

countries while it was lower in Europe and including Russia, and North America (New &

44

Csavas, 1995). The low detection of Salmonella in this study is in concordance with the low

frequency of Salmonella detected clinically

5.2.5 Vibrio spp.

All fish samples examined in this study were found negative for the presence of Vibrio species

opposite results obtained by (Elhadi et al., 2004) and contrast (Yücel & BALCI, 2010).

According to the regulations of International Association of Microbiology Society, fresh and

frozen fish should possess neither Vibrio spp. nor Salmonella spp. The investigated frozen

samples were of good quality as all the samples were free from these pathogenic

microorganisms.

5.3 Questionnaire analysis

Non of interviewed fish farmers has consulted a veterinary and that necessitate the need of

increasing the awareness among farmers. Although only one farm admitted the use of

antibiotics for treatment of fish all farms showed positive samples for residues and this would

make routine measurements of these farm a primary.

47

Chapter VI

Conclusions and

Recommendations

48

Chapter VI

Conclusions and recommendations

6.1 Conclusions

This study evaluated antibiotic residues and bacteriological quality of sold fish and there

is no previous studies or data related to the findings of this study. Therefore, results

obtained in the present study could not be compared with any local data.

The followings could be concluded from the results of this study:

1. The study revealed that 52% of examined fish samples contained antibiotic

belonging to the Aminoglycosides group.

2. Of the 100 fish samples, only one was positive for Tetracyclines.

3. All samples were completely negative for β-Lactams and Macrolides.

4. Antibiotic residues detected in higher frequency in farmed fish than from other

sources. It seems that frozen and fresh caught fish are the safest sources.

5. The percentage of samples that failed to comply with microbiological quality

standards was 39% of all fish samples.

6. Total Plate Count; 4% (4/100) (≥ 106 CFU/g), this percentage was indicator for

poor microbiological quality.

7. Total Coliform bacteria; 39% (39 samples) and S. aureus; 13% (13 samples).

Their presence as indicators for fecal contamination by human or animals.

8. Presence of pathogenic bacteria such as Salmonella spp. (1%) represent health

risks.

9. The high number of contaminated farmed fish could pose a risk for human health

after consumption of undercooked fish and proper cooking of fish is encouraged

to avoid foodborne illness.

10. Additional risk originates from the possibility of cross-contamination.

49

6.2 Recommendations

In spite of the fact that antibiotics will likely always be needed in the operation of fish-rearing

facilities, these compounds must be used with care in order to minimize risks to humans and

the environment. As well as microbial loads should not exceed the recommended levels. In light

of the results of this study and from the accumulating literature, the followings are

recommended.

1. Additional studies should be done investigating the relation between inappropriate

use of antibiotics in aquaculture, and antibiotic residues and antibiotic resistant

pathogens.

2. We recommend strict control by monitoring the usage of antibiotics on fish farms, and

observation of withdrawal period.

3. Farmers who are responsible for the use of antibiotic should receive clear

instructions through the supervision of veterinary authorities and veterinarians.

4. To minimize the use of antimicrobials, it is necessary to apply preventive measures for

disease prevention through improving hygienic and environmental conditions under

which fish are grown and developing vaccination programs for farm raised fish.

5. There is a need for more data on the occurrence of residues of other antimicrobials

(other than those investigated in this study) in aquaculture products from different

production types.

6. More researches are needed to provide data regarding the usage of different

antimicrobials in different types of foods of animal origin and the occurrence of residues

of various antimicrobials in these types.

7. It is recommended to make training on sanitation procedures and programs for fish

worker and this may reduce the presence of pathogens.

8. Improvements in handling and processing are needed to minimize the prevalence of

pathogenic bacteria.

9. Further studies are needed to evaluate the microbial quality of other fish species from

the Gaza strip markets.

10. Regular bacteriological analysis of different fish types must be carried out on a regular

basis to maximize the chances of detecting contamination and pathogens.

71

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72

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84

Annexes Annex 1

االستبانة باللغة العربية

موضوع متبقيات املضادات الحيوية والجودة امليكروبية لألسماك واملقدمة هذه املقابلة تتناول

غزة.-كجزء من متطلبات نيل درجة املاجستير في العلوم الحياتية/ الجامعة اإلسالمية

كل املعلومات التي ستنتج من هذه املقابلة ستستخدم ألغراض البحث العلمي فقط ولن يذكر اسم

املين فيها اال بإذن خطي مسبق.املزرعة وال إدارتها أو الع

املنطقة: املدينة:

عدد األحواض : املزرعة: اسم

ية لألحواض:الكثافة السمك :للمزرعةاملساحة الكلية

مالحظات:

87

ال نعم برجاء اإلجابة على األسئلة التالية بنعم أو بال

هل تستخدم املضادات الحيوية لعالج األسماك؟ .0

هل تستخدم املضادات الحيوية لوقاية األسماك؟ .5

هل لديك علم بطبيعة كل األدوية املستخدمة للعالج أثناء التربية؟ .0

في جسم األسماك له تأثير سلبي على صحة االنسان؟ هل تعتقد ان الدواء .4

هل تعرف أي تعليمات او إرشادات غير الجرعة الستخدام الدواء؟ .2

املستخدمة؟ هل لديك معرفه بفتره السماح لألدوية .0

ل من شفاء األسماك املريضة؟ .7 عج هل تعتقد ان استخدام جرعه مضاعفه من األدوية ت

من مضاد حيوي في فتره عالج واحده؟ هل تستخدم أكثر .8

إذا كانت اإلجابة "نعم" برجاء حدد غير املضادات الحيوية؟ أخري هل تستخدم أي ادويه .3

_____________________________________________________________________________

ك حدد تل "نعم"كانت اإلجابة إذا باألمراض؟ تكثر فيها إصابة األسماك /موسم هل توجد فتره .01

الفترة ...................................

العالج؟ فتره أثناءهل تقوم ببيع األسماك .00

هل لديك ترخيص للمزرعة؟ .05

88

شاكرين لكم حسن تعاونكم

منشط للنمو وقاية عالج الحيوية؟ ما هو سبب استخدام املضادات .00

مكان آخر املزرعة لبيت ا الدواء؟ أين تقوم بتخزين .04

ماء األحواض مدمج مع األعالف الدواء؟ما هي طريقه إعطاء .02

؟من يصف لك الدواء عند وجود أسماك مصابة .00

أكثر يومين يوم متي تتوقف عن إعطاء الدواء قبل التسويق؟ .07

من هي الجهة الرقابية؟ .08

ال نعم السمكية؟هل لديكم أي تواصل مع اإلدارة العامة للثروة .03

مساعدة واستشارة رقابة إشراف ما طبيعة التواصل إن وجد؟ .51

ما هي أنواع األسماك املستزرعة؟ .50

ما هو النوع األكثر استهالكا من قبل املواطنين؟ .55

ما هو مصدر املياه املستخدمة في االستزراع؟ .50

؟األحواضكم مره يتم تغير وتجديد مياه .54

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Annex 2

An English version questionnaire This interview is about antimicrobial residues in fish in Gaza strip

Submitted as part of the requirements for the degree of master of biological

sciences Islamic University-Gaza

All information produced from this interview will be used for scientific research

purposes only and will not mention the name of the farm and its management or

employees except with written permission in advance.

City: Area:

Farms name: Ponds numbers:

Total area of farm: Fish density of ponds:

Notes:

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Subject Yes No

Do you use antimicrobials on fish for Therapy?

Do you use antimicrobials on fish for prevention?

Do you know the nature of all drugs used for treatment during

farming?

Do you believe that drugs in fish body have a negative effect on human

health?

Do you know any instructions apart from dosage instructions or the

used drugs?

Do you know the withdrawal period of used drugs?

Do you think using a double dose of drugs accelerate the healing of

sick fishes?

Do you use more than one antimicrobial in a single treatment?

Do you use medication other than antibiotics?

Mention if present.

Is there a period of increasing the incidence of diseases? • If yes,

determine the period.

Do you retail chickens during treatment periods?

Do you have a license for the farm?

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Enhancement Prevention Treatment What is the reason for using antimicrobials?

Elsewhere House Farm Where do you store drugs? Feed Pond water How do you administer medication?

Never Sometim

es Always Do you consult a veterinarian to describe medications

More two day Would you cease giving drugs before marketing? Do you have any connection with Public Administration of fish

wealth? What is the nature of the connection ?

What are the types of farmed fish?

What is the most consumed type by the inhabitants?

What is the source of water used in farming?

How often do water ponds change and renew?