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?