بسم اهللا الرحمن الرحيم

PREVALENCE OF ESCHERICHIA COLI,

SALMONELLA AND STAPHYLOCOCCUS AUREUS IN PROCESSED MEAT IN

KHARTOUM STATE

A dissertation submitted

By Nadia Mahmoud Hamid Hamad

B.V.Sc. University of Alnileen, 2000

In partial Fulfillment of the requirements for the Degree of M.Sc. by courses and research

Supervisor Prof. Mohammed Taha Shigidi

Department of Microbiology Faculty of Veterinary Medicine

University of Khartoum

2010

i

بسم اهللا الرحمن الرحيم

:قال تعالى

ما إال لنا علم ال سبحانك قالوا{ } الحكيم العليم أنت إنك علمتنا

صدق اهللا العظيم البقرة سورة

ii

PREFACE

This work has been carried out in the

Department of Microbiology, Faculty of

Veterinary Medicine, University of Khartoum,

under the Guidance and supervision of Prof.

Mohamed Taha Shigidi.

iii

To my Father

& my Mother

iv

 

I would like to express my thanks to my supervisor

Prof. Mohamed Taha Shigidi for his guidance,

advice and encouragement.

Great appreciation to the National Health

Laboratory, Department of Food and Water for

permission to use the facilities in the department

and for the invaluable assistance.

My deep thanks are due to A. Zoohor Hamid, My

thanks also go to Hala Hassan, Seham Mustafa,

Dr. Nada Abdo Alagallel and all my friends and

colleagues for their great cooperation and support.

v

Table of Contents

Pageاالية القرانية iPreface iiDedication iiiAcknowledgements ivTable of Contents v-viiList of Tables viiiList of Figures ixAbstract (English) x-xiAbstract (Arabic) xii-xiiiIntroduction 1-3

Chapter One: Literature Review

1.1. Pathogenic Bacteria in Food 41.2. Meat Contamination 61.2.1. Contamination of minced meat by some

pathogenic bacteria 9

1.2.2.Contamination of Burger and Sausage by Some Pathogenic Bacteria

11

1.3. Pathogenic bacteria in processed meat 121.3.1. Pathogenic Escherichia coli 121.3.2. Salmonella 161.3.3. Staphylococcus aureus 181.4.Food Poisoning 211.5.Meat Hygiene and control of pathogens 26

Chapter Two: Materials & Methods 2.1. Methods of Sterilization 302.1.1. Dry Heat 302.1.1.1.Red Heat 302.1.1.2.Hot air Oven 302.1.2. Moist Heat 302.1.2.1.Autoclaving 302.1.3. Filtration 302.1.4. Disinfection of media preparation room 30

vi

2.2. Diluents and Solutions 312.2.1.Buffer Field's Phosphate Buffer 312.2.2. Physiologyical Saline 312.2.3.Rabbit Plasma 312.2.4. Potassium tellurite Solution 312.2.5. Iodine-Iodide Solution 312.2.6. Egg Yolk Emulsion 312.2.7. Urea Solution 322.3. Cultural Media 322.3.1. Solid Media 322.3.1.1. Baird –Parker Medium (Oxoid Code-CM275) 322.3.1.2. Xylose Lysine Deoxycholate (Mast code-DM

23OD) 32

2.3.1.3. Brilliant green agar 332.3.1.4.Nutrient agar (Oxoid code-CM3) 332.3.1.5. Eosin methylene blue agar(Mast code-DM133D) 332.3.1.6.Urea agar base (Oxoid Code-CM 0053) 332.3.1.7.Triple sugar iron Agar (Oxoid code –CM 227) 342.3.2. Liquid Media 342.3.2.1. EC medium (Difco code- 135581 XA) 342.3.2.2. Selenite cystine (Oxoid code-CM 699) 342.3.2.3. Tetrathionate Base Broth (Mast code-DM 2195) 352.3.2.4.Birn heart Infusion (Oxoid code-CM 225) 352.3.2.5.Lauryle sulfate tryptose broth (Oxoid code-

CM0451) 35

2.3.2.6. Peptone water (Oxoid code-CM9R4) 352.3.2.7. Methyl red and Voges Prokauer medium (Oxoid

code-CM 43R 26) 35

2.4.Reagents 362.4.1. Kovac’s reagent 362.4.2. Vages- Proskauer test Reagent 362.5. Sampling 362.5.1. Description of samples 362.5.1.1. Definition of meat 362.5.1.2.Minced Meat 372.5.1.3.Sausage 372.5.1.4. Beef burger 372.5.2.Sources of samples 37

vii

2.5.3. Collection of samples 37 2.5.4. Transportation of collected samples 382.5.5. Preparation of the samples 382.6. Cultural Methods 382.6.1. Method for Presumptive E.coli Detection 382.6.2. Method for Salmonella isolation and Identification 392.6.3. Method for isolation and emumeration of

Staphylocossus. aureus 39

2.6.4. General Examination 402.6.5. Primary testes 40 2.6.5.1. Gram staining & Microscopic Examination 40 2.6.5.2. Sugars fermentation test 412.6.6. Secondary tests 412.6.1. Urease activity test 412.6.6.2. Triple sugar iron (TSI) test 412.6.6.3. Voges – proskauer (v.p) test 412.6.6.4.Indole test 422.6.6.5. Tube coagulase test 422.6.7. Serological test 42

Chapter Three: Results 3.1. Prevalence of different species 433.1.1. Potentially Pathogenic E. coli 433.1.2. Salmonella 443.1.3. Staph. aureus 44

Chapter Four: Discussion Discussion 50Conclusion & Recommendations 56References 57-68

viii

List of Tables

Table Title Page

(1) Shows the prevalence rates of E.coli in the different

meat products

45

(2) Shows the prevalence rates of Salmonella in the

different meat products

45

(3) Shows the prevalence rates of staph.aureus in the

different meat products

46

(4) No. of different bacterial isolates from different meat

products

46

ix

List of Figures

Figure Title Page

(1) Shows the prevalence rates of E.coli in the

different meat products

47

(2) Shows the prevalence rates of Salmonella in

the different meat products

48

(3) Shows the prevalence rates of staph.aureus in

the different meat products

49

x

Abstract Meat and meat products (minced meat, sausage, and burger,

koffta,…) are highly perishable and easily spoiled and

contaminated by bacterial from different sources of contamination

before, during and after processing, and soon become unfit and

possibly dangerous to health. Inspite of increasing consumption of

these products there are no enough studies to assess the level of

contamination with some important pathogenic bacteria which

cause food poisoning.

The aim of this study was to determine the prevalence of E.coli,

Salmonella and staphylococcus.aureus in minced meat, sausages

and beef burgers in Khartoum State. These bacteria are regarded

as the most common causes of poisoning in Sudan.

In this study, some important potentially pathogenic or pathogenic

bacteria which cause food poisoning were isolated, and identified

their prevalence rates in 75 samples of different types of frozen

processed meat products (minced meat, Sausage, Burger) ready

to consumption as a final product was determined. Isolation and

identification of bacteria were carried out according to the

techniques recommended by the International Organization for

Standardization (ISO) which are adapted by Sudanese Standards

and Metrology Organization (SSMO). Identification was based

mainly on morphology and staining reaction and biochemical and

serological tests.

The samples were collected randomly from different factories of

meat processing in Khartoum, Khartoum North and Omdurman,

during the period from April 2007 to February 2008.

xi

E.coli detected by using eosin-methylene blue agar (EMB) as a

selective medium and confirmed by biochemical tests. For

detection Salmonella two selective mediam; xylose lysine deoxy-

cholat agar (XLD) and brilliant green phenol red agar (BGA) and

confirmed by biochemical and serological tests. Stayphylococcus.

aureus detected by using selective Baird-Parker medium and

confirmed by tube coagulase test.

The study showed that the examined specimens contained a high

contamination rate of the above potentially pathogenic or

pathogenic bacteria which reduces the quality of the meat products

and constitutes a health hazard.

Generally the prevalence rate of S.aureus recorded (53.3%) out of

all samples, followed by E.coli with prevalence rate (28%), and

then Salmonella recorded (21%).

The results showed that all samples in this study had

appreciateble rate of bacterial contamination. Sausage had the

highest count rate with S. aureus (72%) followed by adetecion rate

of (40%) with E.coli in burger samples (28%) detection rate for

Salmonellla from minced meat product.

The high prevalence rates of pathogenic and potentially

pathogenic bacteria obtained in this study indicates that hygiene

was poor during processing. Most working staff in the meat

factories may not receive any public health education or training

courses. Poor personal hygiene was obvious among the workers

and hygiene measures which are applied in these factories were

poor. Workers were not asked to use hair covers or appropriate

gloves during handling and processing, which may constitute

public health hazarded for the consumers.

xii

 ملخص البحث

 

من األطعمة سريعة التلف ...) ،فتةاللحم المفرومة، السجق، البيرقر، الك( اللحوم ومنتجاتها

والتى تتلوث بسهولة بكثير من مصادر التلوث المختلفة قبل وأثناء وبعد عملية التصنيع، ومن

د لهذه ورغم اإلستهالك المتزاي. لإلستهالك ومصدر خطر على الصحة اسبةثم تصبح غير من

المنتجات ال توجد دراسات كافية لقياس مستوى تلوثها ببعض من أهم الكائنات الممرضة

.المسببة للتسمم الغذائي

و السامونيال الهدف من هذه الدراسة هو تحديد نسبة إنتشار بكتريا االشريشيا القولونية

رقر فى والية والمكورات العنقودية الذهبية فى كل من اللحمة المفرومة والسجق والبي

.والتى تعتبر من أهم األجناس المسببة للتسمم الغذائى فى السودان. الخرطوم

م مستوالتى تسبب الشار بعض أنواع البكتريا الممرضة فى هذه الدراسة تم تحديد نسبة إنت

لحمة مفرومة، ( نوع من أنواع اللحوم المصنعة 75الغذائى، ومن ثم عزلها من حوالى

عة فى الخرطوم ن غالبية مصانع إنتاج اللحوم الموزلتى أخذت عشوائيا موا) سجق، بيرقر

فى الفترة بين ابريل والخرطوم بحرى وامدرمان فى صورة منتج نهائى مجمد مقدم للمستهلك

.م2008م وإلى فبراير 2007

والتى وضعت (ISO)والتعرف عليها الطرق العالمية القياسية استخدم فى عزل هذه البكتريا

وتم التعرف عليها عن طريق .)SSOM(من قبل الهيئة السودانية للمواصفات والمقاييس

.والسيرولوجية لهذه األجناسدراسة الخواص الشكلية والتفاعالت الصبغية والبايوكيميائية

 لعزل وتحديد االشريشيا تم إستعمال وسط ال (EMB) كوسط إنتقائى وأكدت بواسطة

) BGA( و) XLD(إستخدم لعزل السالمونيال وسطين انتقائيين هما . البيوكيمائية االختبارات

واكيدت إيضا باإلختبارات البيوكيميائية والسيرولوجية، أما لعزل البكتريا العنقودية الذهبية

.و تم تاكيدها بإستخدام إختبار التخثر االنبوبى) BP(إستخدم الوسط االنتقالى

ل تلوث عالى للحوم المصنعة بالبكتريا المفسدة للطعام والتى قد تسبب عكست هذه الدراسة معد

التسمم الغذائى فى حالة وجود الظروف المناسبة لذلك والتى تؤدى لتقليل جودة المنتج و

.بالتالى تشكل مخاطر على الصحة العامة

اصل جميع العينات من %) 53.3(عموما النسبة المئوية التى سجلتها البكتريا العنقودية الذهبية

.سجلتها السامونيال%) 21(ثم %) 28(تليها االشريشيا القولونية بنسبة إنتشار

أوضحت هذه الدراسة أن جميع أنواع العينات التى تم فحصها تحتوى على نسبة عالية من هذه

البكتريا الممرضة حيث سجل السجق أكبر نسبة من البكتريا العنقودية بنسبة تصل إلى

xiii

من بكتريا )%28(من البكتريا القولونية ثم نسبة )%40(يليه البيرقر حيث سجل ) 72%(

.السالمونيال تم عزلها من اللحمة المفرومة

معظم ه البكتريا الممرضة تدعم اإلعتقاد بأنهذه النسب المرتفعة التى تحصل عليها من هذ

أو تدريبية فى كيفية التعامل العاملين فى مصانع إنتاج اللحوم لم يتلقوا أى كورسات تعليمية

الصحى مع هذه المنتجات االستهالكية أيضا أتضح تدنى المستوى الصحى الشخصى لهؤالء

العمال حيث كانوا ال يرتدون اغطية الرأس والقفازات المناسبة المعده لهذا الغرض وهذا كله

 . يمثل خطر صحى داهم بالنسبة للمستهلك

1

Introduction

Meat and meat products are concentrated sources of high quality

protein and their essential amino acids content usually

compensate for deficiencies in diets made mainly of cereal and

other vegetable proteins. They supply easily absorbed iron and

assist in the absorption of iron from other foods as well as zinc,

and are rich sources of some of the B- vitamins. By providing such

nutrients, meat consumption can alleviate common nutritional

deficiencies (Bender, 1992).

(Sharmoot) the local name for sun-dried meat was the earliest

processed meat known in this country (Mohamed, 1982). Since old

times people have known meat processing and preservation in a

conventional way. Due to increased meat production and

appearance of meat industry, now there are various forms of

processed meat products such as minced meat, sausages, beef

burger and others. So care and effort must be taken to provide

safe and clean products to the consumers.

Food spoilage and food poisoning by microbes have been known

since 1880 (Jay, 2000). Chemicals, heavy metals, parasites, fungi,

viruses and bacteria can cause foodborne illness (Cliver, 1990) .

Meat and its products are highly perishable and spoil easily, and

soon become unfit to eat and possibly dangerous to health through

microbial growth, chemical changes and breakdown by

endogenous enzymes (Judge,M.D,Aberle,E.D,Forest ,J.c, Hedrick,

M.B and Merkel, R.A,1990).

There are many factors affecting the shelf life of meat and meat

products, that include extrinsic factors such as temperature of

2

preservation, relative humidity, the availability of oxygen, and

physical state of meat.

The other intrinsic factors such as water activity, pH, oxidation-

reduction potential, the presence or absence of inhibitory

substances and protective tissues, and the nutritive value of meat

should be considered to avoid spoilage deterioration and spoilage

of meat (Judge et al., 1990; Lawrie, 1990).

Sudan has a huge livestock population, estimated as more than

one hundred and twenty million heads. Therefore, modern aspects

of animal production efficiency based on recent scientific

developments must be considered, especially slaughter and

processing techniques with good control of sanitation and hygiene.

These will result in greater yields and higher profits, and would

also provide incentives for increased production (FAO, 2000).

Microbial contamination of food is an increasing public health

problem, enhanced by the modern necessity of consuming food

outside home. Frequently, the raw materials utilized to prepare

food contain, live microorganisms and some of them may be able

to cause disease in susceptible hosts. Improper manipulation and

cross contamination during industrial processing or improper

handling by the consumer also contribute to spread of dangerous

microorganisms (Gustavo,F.,Miriam,T., Faola,N. and Guillermo,

2004).

Lawrie (1990) indicated that the increasing pressure of the world

population, and the need to raise living standards, have made the

production of more and better meat a necessity. Judge et al.

(1990) also expected that the pressure of increasing world

population and associated ecological problems dictate that a

higher efficiency of food production must take high priority, and

3

that every aspect of meat production from conception to

consumption must be carefully evaluated in seeking ways to

improve efficiency.

There are many constraints and problems facing meat production

in most of the developing countries. According to Bender (1992)

these include inadequate feeding and poor management of

animals to which must be added animal health problem, lack of

skilled and trained manpower, problems arising from land tenure,

lack of financial resources, poor infrastructure and roads, poor

health care, unhygienic conditions in slaughter, poor management

of all aspects of meat production and storage.

In Sudan, Salih and Ibrahim (1975) discussed the relation between

the spoilage of meat, its bacteriological standards and food

poisoning. Contamination of human origin calls for better

safeguards required to protect the consumer. Other bacteriological

studies in processed meat (Fatima, 1982, 1990) were made, but

additional research is needed to identify the level of contamination

at different stages of the processing operation.

The objective of this study is to: (1) assess the level of

contamination of minced meat, sausages and beef burgers with

the potentially pathogenic Escherichia coli, and pathogenic

staphylococcus aureus and Salmonella in Khartoum State which

constitute a public health hazard in such processed meat products.

4

Chapter One Literature Review

Meat is a highly perishable product and soon becomes unfit to eat

and possibly dangerous to health through microbial growth so

Contamination of meat by microbial agent is a subject of significant

importance (Bender, 1992). After becoming contaminated, meat

and meat products provide an excellent environment for growth of

bacteria.

Bacterial contamination and growth is a problem because it may

result in food borne illness. Food borne infectious diseases have

emerged as an important public health problem in many countries

in the last decade (Motarjemi and Kaferstein, 1997).

1.1. Pathogenic Bacteria in Food: Most species of bacteria are relatively harmless or even beneficial

to man, and only a small number causes illness if they are present

in food. The most important of these are Bacillus cereus, Bacillus

subtillis, Salmonella, Campylobacter, Escherichia coli, Clostridium

perfringens, Cl. Botullinum, Staphylococcus aureus, Listeria and

Yersinia (POST, 1997).

Raw beef and beef products could inevitably contain pathogenic

microorganisms (Nichlos and de Louvois, 1995). Besides various

Gram-negative (Escherichia spp., Enterobacter spp., Yersinia

spp., and Pseudomonas spp.) and Gram-positive bacteria

(Bacillus spp., Micrococcus spp. and Lactobacillus spp.),

psychrotrophic moulds (Aspergillus spp., Cladosporium spp.,

Geotrichum spp., Mucor spp . and Rhizopus spp.) are frequently

5

isolated from the meat surface (Polster ,M.D,Hartiova,I.

Kralikova,1985).

Gracey (1981) reported that, the organisms responsible for food

poisoning by infection were Salmonellae, Escherichia coli and

Vibrio parahaemolyticus. Those responsible for poisoning by toxin

production included Staph.aureus, Clostridium perfringens,

Cl.botulinum, Bacillus cereus and Streptococci. Other bacteria

occasionally which may cause outbreaks of food poisoning,

included, Streptococci, Proteus, Pseudomonas, Providencia,

Citrobacter, Aeromanas Hydrophila, Yersinia enteracolitica,

Campylobacter, and Shigella flexineri.

Fatima (1982), emphasized that pathogenic bacteria found in

processed meat were Salmonella spp, Cl perfringens, Staph.

aureus and E. coli.

Salmonella.spp and Staph.aureus are the most commonly

implicated organisms in food borne illnesses (Mead, P.S,Slutsker,

and Dietz, 1999).

Food Safety Inspection Services (U.S.A.,1997) reported that,

pathogenic bacteria related to meat and their products were

B.cereus, Cl. botulinum, Cl.perfringens, E.coli, Salmonellae, Staph.

aureus and Y. enterocolitica.

Hussein (1975) isolated from fresh sample; Staphylococcus

epidermidis, Micrococcus spp, E.coli, Proteus spp, Aeromonas

spp, Pseudomonas spp, and Acromobacter spp. No Salmonella

or coagulase positive Staphylococci were isolated.

Fedral register (U.S.A., 1999) reported that E.coli 0157: H7 had

been linked to or found in ground beef that caused food-borne

illness.

6

In Sudan meat–borne bacteria detected were Staph.aureus,

Bacillus cereus, E. coli, Salmonella spp, Klebsiella spp and proteus

spp (Hussein, 1987; Mohamed, 2000; Elhussein, 2003).

1.2. Meat Contamination: Raw materials, as well as final meat products are exposed to a

high risk of microbial contamination at the time of their production,

processing, storage and distribution. Chemical composition of

food, properties of the outside environment and specific growth

requirements determine the type of microorganisms and the

course of physical and chemical reactions in the contaminated

food (Lacia kova et al, 2004).

Meat is an ideal environment for the growth of microorganisms

particularly bacteria (Frazier, 1967; Forsythe and Hayes, 1998).

Microorganisms can multiply readily on the cut surfaces and

exposed surfaces which become easily contaminated after

slaughter and during and after dressing and butchering, although

the microbial count of the interior of the meat usually remains

much lower (Harrigan, 1998).

Center for Disease Control and Prevention (2000) reported that

food of animal origin such as poultry; eggs and beef are the main

sources of food borne illnesses. Raw beef, salami and sausages

have all been associated with Salmonella outbreaks in United

States of America.

Banwart (1981) reported that so as to control contamination,

microbial load on the food must be kept as low as possible. It is

important to know the main sources of contamination. Hence,

contamination of meat product was extensively studied.

7

In general, it can be concluded that meat can become

contaminated if the meat is eaten from an animal that had

septicemia, if infectious lesions remain on sites used for meat, if

edible organs are infected or inedible infected organs leaked

bacteria on surfaces or if cross-contamination occurs from animal

faeces (Bryan, 1978).

The microbiology of meat is highly dependent on the condition

under which animals are reared, slaughtered and processed

(Brown, 1982). The only difference between sterile food and

natural food is the bacterial population (Wafa,1999).

Vanderzant and Neckelaon (1969) and Frazier and westhoff

(1978,1989), it was found that the main sources of contamination

of meat were the soil and drinking water (Frazier, 1967) also

(Banwart, 1981) and air (Jay, 1970; Mackenzie and Bain, 1976).

The microbial flora that grows on refrigerated beef is composed

mainly of soil organisms which contaminate the hide and hoof of

the animal (Banwart, 1981). And the major contamination of meat

during processing is from skin or hides (ICMSF, 1980). Any

contaminating bacteria on the knife soon will be found on meat in

various parts of the carcase (Frazier, 1967).

The level of microbial contamination on meat was influenced by

the level of carcase contamination at boning and by boning

process it self. Carase contamination was the major determinant of

microbiological quality, a some of more than 70% of carcase had

microbial counts greater than 103/cm2. Cutting boards were a

major source for microbial dissemination during boning

(Widders,P.R., Coates, K.J.,Warener, S.W., Beattie, J.C.,Margan.

R and Hickey, M.W;1994).

8

During transport form farm to the slaughter house livestock are

herded together and frightened so that cross-contamination occurs

more readily (Forsythe and Hayes, 1998).

Frazier and Westhoff (1988) pointed out that during bleeding,

skinning and cutting the main sources of microorganisms were the

exterior of the animal, intestinal tract, knives, air, hands and

clothes of the workers.

During handling, contamination comes from special equipments

(grinders, sausage stuffers and casing) and ingredients in special

products (spices).

Mohamed (2000) found that spices, additives and machines used

during processing were sources of contamination to the meat

during processing.

Wagner (2000) reported that meat would be contaminated by

contact with hides, feet, skin, stomach and intestinal contents and

clothing of personnel, water used for washing carcase,

equipments, and even air in the processing and storage areas.

Meats when put in utensils, some or all microorganisms on the

surface of these meats will contaminate the contact surface of the

utensils. Fresh meats put in the same utensils will inevitably pick

up some of these microorganisms then become contaminated

(Jay, 2000).

Hussien (1971) isolated bacterial contaminants of fresh meat from

the gastro-intestinal tract and hides of the slaughtered animals and

from the water and air deposits.

Wilson (1981) also reported that contamination of processed meat

may occur during preparation stage.

Food Safety and Inspection Service (U.S.A., 1997) pointed out that

microbiological contamination of carcasses occurred during

9

skinning, evisceration, washing and chilling, and also during the

processing steps which included handling, grinding boning and

addition of ingredients (spices, bread, groundnuts).

The main sources of contamination is humans (handlers

contaminate food via manual contact or via the respiratory tract by

coughing and sneezing), and contamination occurs after heat

treatment of the food.

Foods such as raw meat, sausages, raw milk,cheese,

contaminations from animal origins are more frequent and due to

animals carriage or to infections (e.g mastitis) (Yvesleloir et al.,

2003).

There are many critical points during the period of production

through slaughter and in further processing. The delivery of clean,

unsoiled animals for slaughter is one of clearest examples.

However, meat hygiene and meat inspection must be improved to

meet effectively those challenges from new animal's production

methods and disease patterns (Johnson, 1994).

1.2.1. Contamination of minced meat by some pathogenic bacteria: Narsimha and Ramesh (1988) proved that comminuted meat

provided an ideal environment for the growth of bacteria.

Federal register U.S.A (1999) reported that, raw ground beef

products presented a significant Public health risk, because they

were frequently consumed after preparation that did not destroy

the organisms that had been introduced below the product’s

surface by chopping or grinding.

Although beef in general may be contaminated , ground meat is

a special concern because grinding combines meat from different

10

animals and transfers bacteria from the meat’s surface to it’s

interior (MFMER,2006).

Minced beef is a product with large and varied microbial profile

which frequently included a number of potentially pathogenic

bacteria. More handling especially grinding and mixing, means a

greater like hood of contamination by bacteria such as Salmonella,

Campylobacter , Listeria , and E.coli O157:H7 ( Mary and Scottie,

1998). These bacteria resulted in rapid spoilage, with shorter-life,

leading to economic losses (Narasimha Rao and Ramesh, 1988).

E. coli O157:H7 was first described as a cause of human illness

and associated with under cooked ground beef in 1982 (Riley,

L.W, Remis, R.S., and Helgerson, S.D,1983).

Food of bovine origin, particularly ground beef, are common

causes of sporadic infections and outbreaks of E. coli O157

(Griffin,P., Mead, P., and Sivaplasingam, 2002).

Raw ground beef previously has been implicated as a vehicle for

transmission of Salmonella (Fontaine, R.E, Arnon,S., and Martin,

1978), the prevalence of Salmonella a beef ranges from 1% for

raw beef carcasses to 5%-7% for ground beef (U.S. Department of

Agriculture, food Safety and Inspection Service, 1994).

Staphylococcus, Escherichia and other bacteria were isolated from

minced meat (Gomes,T.M,Motarjemi,Y,Miyagawa,S.,Kaferstein,

F.K, Stohr.1997).

Beckers et al. (1986) and Cloak et al. (1999) isolated Salmonella

from minced beef samples.

Elgohary (1994) isolated Staph aureus (38.3%), E.coli (75%) from

minced meat samples. Vorstar et al., 1994 also isolated Staph

aureus from (23.4%) ground beef minced samples.

11

The coagulase positive staph count of frozen minced beef must be

less than 103 bacteria per gram with the absence of Salmonella

and E. coli (SSMO, 2001).

1.2.2.Contamination of Burger and Sausage by Some

Pathogenic Bacteria: Sausage is a processed meat that might be contaminated during

preparation (Wilson,1981; Food Safety and Inspection U.S.A,

1997).

Herschdoerfer (1968) explained that fresh sausage was largely

retailed in non-frozen condition and consequently highly vulnerable

to bacteria spoilage and that the rate of deterioration depended

primarily upon the initial contamination of the raw material

sanitation during processing, temperature history and addition of

permitted preservatives.

Jay (1986) reported that sausages is usually contaminated with

more varied flora than most other processed meat due to different

seasoning agents employed.

Meat is the most prominent food for E.coli O157:H7 outbreaks.

This strain grows in the intestines of mammals, it is most frequently

spread via contaminated animal meat (Teresa, 1999). Outbreaks

were linked to contaminated hamburgers and the illness earned

the nick name “hamburger disease”.

Consumption of undercooked meats and mainly hamburgers has

been strongly implicated in hemorrhagic uremic colitis and

hemolytic uremic syndrome (Yu and Bruno,1996; Venkateswaran,

K.,Kamijoh,Y.,Ohashi, E and Nakanishi.,1997).

Raw sausages, burgers, ground beef and other undercooked raw

meat products will often contain various combinations of cereal,

rusk, salt and spices (Harrigan, 1998).

12

Spices and other additives were found to be a source of

contamination. This because there was no previous treatment of

the spices to eliminate their microbial content (Frazier and Westoff,

1988). It contents a significant numbers of coliforms, staphylococci,

and lactobacilli, and heating at 800C for 1h lowered the counts

approximately to 10-fold for all types of bacteria in sausages

(Verma , M.M, Ledward, D.A, and Lawrie, 1987).

Asha (1991) isolated E. coli from sausage samples, and the

presence of these organisms in meat was taken as indicator of

meat contamination with faeces (Harrigen, 1998).

Mohamed (1991), Mohamed (1997) isolated Staph.aureus from

fresh meat, fresh sausage and fresh beef burger samples.

Isolation of Salmonellae from sausages beef was recorded by

Skjerve and Olsvik (1991) and Coleman et al., (1995).

Raw meat and raw meat products frequently harbor Salmonellae

where as and the low-level contamination of food with S. aureus is

unavoidable (Wallace, R.B, Doebbling, B.N and Last, 1998).

Coagulase positive Staph count of frozen uncooked meat (burger,

sausages, koffta…) must be less than 103 bacteria per gram with

the absences of Salmonella and E.coli (SSMO, 2001).

1.3.Pathogenic bacteria in processed meat: 1.3.1.Pathogenic Escherichia coli: The organism originally called Bacterium coli, was isolated from

the faces of infants by Teodor Escherich over a century ago, in

1920, the organism was renamed Escherichia coli (Willshaw,

G.A,Smith, R,Roberts,D.,Thirlwell,J.,Cheasty,T.and Rowe,1993).

E. coli is member of the coliforms that live in the intestinal tract of

healthy people and animals. Most of them are harmless and play

13

an essential role in absorbing certain vitamins, but few strains of

E.coli are responsible for serious food-borne infections (MFMER,

2006).

Escherichia coli are members of family Enterobacteriaceae, this

organism is Gram-negative, facultative anaerobic, motile, non-

spore forming short rods, catalase-positive and oxidase-negative.

Most strains ferment lactose, methyl red positive and Voges-

Proskauer negative and most strains produce indole (ICMSF,

1996).

The major sources of the bacteria in the environment are probably

the feces of infected humans, but there may also be animal's

reservoirs. Feces and untreated water are the most likely sources

for contamination of food (Wagner, 2000).

Members of the coliform and fecal coliform groups are referred to

as indicator organisms since a quantitation of their presence was

used to indicate the potential presence of pathogens in foods. It

was believed that the higher the number of coliforms, the greater

the possibility of pathogenic organisms being present (FAO, 1992).

The regular presence of E.coli in human intestine and feces has

led to tracking the bacterium in nature as an indicator of fecal

pollution and water contamination (ICMSF, 1996; Kenneth, 2008)

and because of the recent involvement of E.coli in several cases of

food poisoning, it is used rather than others as an indicator of

sanitary quality (FAO, 1992).

While most of E.coli strains are a common part of the normal

facultatively anaerobic micro flora of the intestinal tracts of humans

and assumed as friendly, some serotypes like O157:H7 are the

most important food-borne pathogens for humans. The infectious

dose of E.coli O157 is very low less than 102, the organism can be

14

transmitted efficiently not only via contaminated foods but also

from person to person (Doyle, 1991).

Non- pathogenic E.coli strains can be isolated in massive numbers

from the intestines of warm blooded animals. However, many

strains of E.coli are pathogenic to humans to varying degree and

these strains can be divided in to six groups (Olsvik, O.,

Wastesony, Y.Lund, A.and Homes,E,1991).

Over 700 serotypes of E.coli are recognized as a pathogens, E.coli

is best known for its ability to cause intestinal diseases.

Four pathogenic types of E.coli associated with food-borne illness:

entero-pathogenic (EPEC), entero-toxigenic (ETEC), entero-

invasive (EIEC) and entero-haemarrhagic E.coli (E-coli 0157;

EHEC) (Eely, 1997; Robert et al; 1998). Forsythe and Hayes

(1998) added two additional strains: enteroaggregtive E.coli

(EAEC), and verotoxigenic E.coli (VTEC), all theses strains were

grouped on the basis of serotyping of the somatic (O), flagellar (H),

and capsular (K) antigens. Each class falls within a serological sub

group and manifests distinct features in pathogenesis.

E. coli (EPEC) cause sever diarrhea in infants its produce one

more cytotoxins and causes serious problems in developing

countries.

E. coli (EAEC) are associated with persistent diarrhoea in young

children. These strains adhere to the intestinal mucosa and cause

non-bloody diarrhea without invading or causing inflammation,

these strains produce toxins. The E.coli ( ETEC) cause diarrhea in

both infants and adults, the disease is usually known as traveler’s

diarrhoea. These strains produce two enterotoxin: a heat- labile

toxin (LT) and a heat – stable toxin (ST) .ETEC strains have been

15

implicated in out breaks of infection with water and foods (meats

and poultry, milk, and cheese).

E.coli (EIEC) resembles Shigella dysentery and includes a

dysentery-like diarrhoea with fever and they don’t produce LT or

ST toxin. E.coli (EHEC) also produce cytotoxins which give more

severe symptoms. E.coli O157:H7 is the most well- known EHEC

strain. EHEC infections are mostly food or water borne and have

implicated in undercooked ground beef in the United States

associated with hamburgers. E.coli (VTEC) are an important cause

of gastrointestinal illness and children under five years have the

highest incidence of diagnosed infections. (ICMSF, 1996; Forythe

and Hayes, 1998; Kenneth, 2005).

The symptoms of illness typically begin about 2-10 days after the

initial infection. The first signs are fever and sudden abdominal

cramps and bloody diarrhoea may follow. Other symptoms include

nausea, vomiting and a mild fever (Griffin et al; 2002).

Although E.coli is part of the normal flora of the large intestine of

humans and animals, most strains at this site are non-pathogenic

however, some strains can produce enteric, urinary tract and

wound infections as well as food poisoning (Eley,1996).

Intestinally pathogenic E.coli defined as these E.coli strains that

are capable of causing diarrheal disease in man or animals

(ICMSF, 1996).

An estimated 73.50 cases of illness, 2000 hospitalization , and 60

deaths occur in the United States each year as the result of E.coli

O 157 infection; many illnesses of this organism are associated

with ingesting contaminated food or drink (CDC,2001).

16

1.3.2. Salmonella: The genus name was coined by Lignieres in 1900 in honour of Dr.

Salmon’s work. Salmonella typhi, the etiological agent of human

typhoid was discovered in 1885 by Eberth and isolated in 1884 by

Gaffky (Salmon and Smith, 1885). Bud (1874) reported that

typhoid fever was transmitted by water and food.

Gartner first isolated Salmonella from meat that had caused food

poisoning in 1888 (Jay, 2000).

The first laboratory-confirmed outbreak of foodborne Salmonellosis

involved 57 persons who ate meat from a sick cow. S.enteritidis

was isolated from the victim who had not survived and from the

meat and blood of the animal.

Since then, Salmonellae have become recognized as a major

cause of enteric fever and gastroenteritis.

In humans, Salmonella are the cause of two diseases called

Salmonellosis: enteric fever, resulting from bacterial invasion of the

bloodstream, and acute gastroenteritis, resulting from a foodborne

infection (ICMSF, 1996;Kenneth, 2005).

Salmonella is a genus of the family Enterobacteriaceae (Brenner,

1984). Members of the family are characterized as Gram-negative,

rod-shaped bacteria, facultative anaerobic, non-spore forming,

usually motile with peritrichous flagella except S.pullorum and

S.gallinarum. The organisms produce acid and some times gas

from glucose, are usually catalse-positive and oxidase-negative

and reduce nitrates to nitrites and produce hydrogen sulphide.

The methyl-red reaction is positive, the Voges-Proskauer test is

negative and indole is negative.

Most members of this family are found in the intestinal tract of man

and other animals as either pathogens or commensals.Their

17

optimum growth temperature as with food poisoning bacteria is

380C (Forsythe and Hayes, 1998; ICMSF, 1996).

Salmonella genus contains over 2200 strains (termed serovars or

serotypes) according to their O-and H-antigens, bacteria related to

each other both phenotypically and genotypiccally by DNA

sequence. Many serotypes are named after the place where they

were first isolated, e.g.S. Dublin, S. london, other names according

to the disease and affected animal, e.g.S.typhi, S.cholerae-suis, S.

abortus-ovis.Serotypes are further sub-divided by their resistance

to bacterio ophages (phagetypes or lystotypes), antibiotics or

heavy metals; their biochemical characteristics (biovars or

biotypes) or their sensitivity to or production of bacteriocins

(ICMSF, 1996; Kenneth, 2005).

Salmonella enterica is an important agent of food borne illness.

This species is sub-classified into subspecies of which S.enterica

subspecies enterica is the most important for human health

(Kenneth, 2005).

Most cases of Salmonella food poisoning outbreaks are caused by

two serotypes:S.enteritidis and S.typhimurium (Saudi

Epidemiology Bulletin, 2001).

S.typhi and S.paratyphi in meat products are indicate of human

origin and their presence therefore indicate poor personal hygiene

occurred during handling of meat products (Neema , M, Sisai, M

and Berhanu, and Ashe, A.G, 2004).

Salmonellae induce the illness by their death following

multiplication in the host’s gut and their subsequent lysis with the

release of a potent endotoxin ( D’Aoust,1991).

The principal symptoms of salmonellosis are nausea, vomiting,

abdominal pain and diarrhoea which may be preceded by

18

headache, fever and chills with a low mortality rate. The condition

needed for an outbreak is the ingestion of live cells present in a

raw food or a processed food via cross-contamination that leads to

human infection (Wallace et al., 1998).

1.3.3. Staphylococcus aureus: Staphylococcus (meaning grape-like clusters) was first named by

Alexander Ogston, when he described the presence of this

organism in pus taken from human abscesses (ICMFS, 1996).

Denys was the first to associate staphylococcus with food

poisoning in 1894 (Jay, 2000). The role of the organism in food

poisoning was rediscovered in 1930 by Dack. Since then S.aureus

has been demonstrated to be a common and wide spread food

poisoning organism (ICMFS, 1996).

Traditionally, the genus Staphlococcus is subdivided into two

groups of strains on the basis of the coagulase test (based on the

ability to coagulate plasma).

The vast majority of food poisoning strains are Staph. aureus

(coagulase-positive). However, at least one outbreak has been

reported due to Staph.epidemidis (coagulase-negative) which is

normally a present in normal skin as a commensal organism and

does not produce an enterotoxin but when found in food may

reflect poor levels of hygienic practices ( Eley,1996).

Staph.aureus is the type species of the genus Staphylococcus

which occurs as Gram-positive, small, non-spore forming, non-

motile organism, forming irregular clusters of cells like bunches of

grape. These organisms are facultative anaerobes, catalase-

positive and oxidase-negative. Their optimum growth temperature

is around 370C. Staph .aureus is unusual in being able to tolerate

19

low water activity levels and thus it grows in fairly high salt

concentration levels (Omer, 1990; Eley,1996; Forsythe and Hayes,

1998).

The most important source of S.aureus is probably the human

body, the principal reservoir being the nose. Between 30 and 40%

of healthy individuals carry S.aureus and many of these nasal

carriers inevitably also harbor the organism on their hands and

other parts of their body. Many lesions such as boils, carbuncles,

septic cuts are abound with S. aureus whilst another source of the

organism is hair. It is also transmitted to food from a human source

by cross-contamination and from another source such as utensils.

Animals are important sources of organism like milk and carcass

meat (Bryan, 1978; Eley, 1996; Forsy the and Hayes, 1998).

Coagulase-positive staphylococci are characterized by the

production of an extra cellular enzyme, coagulase that convert

fibrinogen in citrated human or rabbit plasma into fibrin, by aiding

an activator present in plasma (Wood, G.D., Richard, C.B,Slack

and John,1992).

All S.aureus strains are coagulase positive (i.e. possess an

enzyme coagulating blood plasma) but only about 30% of strains

are able to produce the enterotxins associated with food poisoning,

six endotoxins, A,B,C1,C2,D and E have been identified.

Types A and D being the most commonly involved with food

poisoning, and are produced during storage. Each enterotoxin

produced has the property of heat resistance (resist boiling for 30

minutes). They are produced when Staph.aureus grows in

carbohydrates and protein food. Enterotoxin A is the most

important cause of food poisoning. (Forsythe and Hayes, 1998,

Jawez and Adel,1990).

20

The diseases caused by staphylococci include acute infections,

such as septicemia, and acute toxemias, such as staphylococcal

food poisoning (ICMSF,1996). After ingestion of the contaminated

food the symptoms appear within 1-6hrs. Food poisoning due to

S.aureus is characterized by nausea, vomiting, abdominal pain

and prostration, often with diarrhoea but with out fever, and the

mortality rate is very low or nil. Recovery is rapid, usually within 2

days (Eley, 1996; Jay, 2000).

S.aureus competes poorly with other bacteria and thus seldom

causes food poisoning in a raw product. The organism is resistant

to drying and having a low water activity also it is very resistant to

salt. The toxins also appear able to survive sterilization and are not

destroyed by cooking (Bergdoll,1989).

Staphylococcal toxin shock syndrome (TSS) is an acute multi

system illness due to infection with S.aureus, it was first described

by Todd et al. (1978), this syndrome is clinically characterized by

sudden onset of fever, hypotension, vomiting, diarrhea, rash and

subsequent desquamation of skin. Case-fatality rate for TSS is in

the range of 1 to 2% (Wallace et al.,1998).

This disease is ordinary not fatal, and it is considered that death

from this disease occurs only when the patient is already weekend

or in a sickly condition when the poisoning occurs (John and

Anthong,1974).

Meat is an excellent medium for staphylococci proliferation and if

the temperature is warm enough only few hours are needed for the

production of effective amounts of enterotoxin. In a bacteriological

study on meat products in the Sudan which included ground meat,

pastorma, beef burger, kofta and sharmoot, Sanousi and Al mahi,

21

1986 isolated several pathogenic bacteria among which the

pathogenic staphylococci were found.

The primary selective isolation of staphylococci from specimens of

a mixed flora is the beginning of a series of confirmatory tests.

Baird-Parker selective and diagnostic medium is mainly used for

coagulase –positive pathogenic species (Baird-Parker, 1962).

1.4.Food Poisoning: A wide variety of diseases can be caused by eating food

contaminated with pathogenic micro-organisms or their products,

by no means all of these diseases can be called food poisoning.

World health organization adopted this definition for food

poisoning: "any disease of an infectious or toxin nature caused by

or thought to be caused by the consumption of food or water"

(Eley, 1996).

There are several differences between foodborne infections and

intoxications. Intoxicating organisms normally grow in food prior to

consumption, which is not always true for infectious organism.

Organisms causing intoxications may be dead or non viable in the

food when consumed; only the toxin need to be present. Organism

causing infections must be alive and viable when food is

consumed .

Infection-causing organisms invade host tissue and symptoms

include headache and fever. Toxins usually don’t causes fever and

act by widely different mechanisms (Wallace et al.,1998).

The different types of food poisoning generally associate

themselves with specific food (e.g.Salmonella with meat products),

there is ample evidence to show that the foods most commonly

22

implicated in food poisoning in general are the various meat

including poultry (Forsythe and Hages,1998).

There are many different types of food poisoning of both microbial

and non-microbial origin. The vast majority of reported case of food

poisoning are bacterial (Eley,1996). Bacterial food poisoning was

first described by Dr. Graether in 1888 (Jay, 1970).

It is customary to divide bacterial food poisoning into the infection

type and toxin type. An infection food poisoning is characterized by

an acute gastroenteritis following ingestion of food in which

multiplication of bacteria has taken place. The ingested viable

bacteria continue to grow within the body to produce the typical

symptoms. This group includes Vibro, Yersinia and Escherichia, as

well as the most common cause of bacterial food poisoning

Salmonella and Campylobacter.

The toxin type (often termed intoxication) in which the enterotoxin,

is present in food having been produced by the bacteria grown in

the food prior to consumption. Bacteria causing toxin poisoning

include Staphylococcus aureus, Clostrisium botulinum,

Cl.perfringens and Bacills cereus (John and Anthony, 1974; Eley,

1996; Forsythe and Hayes, 1998; Wallace et al., 1998).

Improper holding temperature, inadequate cooking and

preparation, contaminated equipment, cross-contamination and

poor personal hygiene are the main factors which contribute to

food poisoning and leading to food borne outbreaks (Robert et al.,

1998).

Diarrhea, vomiting and abdominal pain are probably the most

common symptoms of food poisoning (Eley,1996).

Meat is the most prominent food for E.coli O157:H7 outbreaks.

Outbreaks caused by EPEC,EIEC or ETEC occur very infrequently

23

in developed countries. Enterohaemorrhagic or Verotoxigenic

strains are the most serious E.coli found in food (ICMSF, 1996).

Enterohaemorrhagic E.coli was associated with two outbreak of

hemorrhagic colitis, many subsequent outbreaks, often associated

with eating under cooked ground beef, have been reported

(Doyle,1991).

The infectious dose for most strains of E.coli is high (106-108

orgs/g) although for Enterohaemorrhagic strains like E. coli

O157:H7 it is much lower. Growth of this strain on meat should be

controlled by rapid cooling to less than 70C after slaughter and

processing (Wallace et al , 1998).

To reduce the risk of E.coli infection, ground beef products,

should be cooked thoroughly to internal temperatures of at least

1600F(710C). Using meat thermometers to ensure this temperature

is reached and it’s enough to kill bacteria (CDC,2004).

Staphylococcal food poisoning is generally suspected when a

number of people develop acute predominantly upper

gastrointestinal tract symptoms a few hours after eating some food

items in common. The foods implicated in this poisoning contains

at least 105 S.aureus organisms per gram to produce sufficient

enterotoxin to cause a human disease (Wallace et al;1998).

The six staphylococcal enterotoxins are simple proteins, under the

best conditions entertoxins will become evident within four to six

hrs and when they produced in sufficient amount result in food

poisoning (Pereira et al., 1991).

An important characteristic of the entertoxins is their stability to

wards heat (Frazier and Westhoff,1989). Most food poisoning is

from type A. The temperature range for growth and toxin

production is about 40C to 460C, depending on the food.

24

Staphylococcal enterotoxins (SE) production is optimal in neutral

pH and decreases in acidic pH. Usually, SE production is inhibited

in pH below 5 (Notermans and Heuvelman, 1983).

The presence of large number of staphylococci in any food stuff is

to be considered undesirable , as pointed out by Thatcher and

Ross (1960).

In all cases of staphylococcal food poisoning, the foodstuff or one

of the ingredients, must have been contaminated with an SE-

producing S.aureus strain and was exposed, at least for a while, to

temperature that allowed S. aureus growth. Many different foods

can be a good growth medium for S.aureus, and have been

implicated in staphylococcal food poisoning, including milk, butter,

cheeses, sausages, etc. (Bergdoll, 1989).

The foods that are most involved in this poisoning differ widely

from one country to another; this may be due to differences in the

consumating and food habits in each of the countries (Yvesleloir,

Florence, B and Michel, 2003).

The infective dose required to induce staphylococcal food

poisoning in humans is estimated to be around 0.1µg and it may

vary with patient sensitivity (Evenson, M.L, Hinds, M.W, Bernstein

and Bergdoll, M.S,1988).

Salmonellosis is the classical form of microbial food poisoning in

which human beings may be implicated as the source of out

breaks.

A number of foodstuff have been associated with salmonellosis,

typically meat products, eggs, raw milk and dairy products, and

any foods that have undergone faecal contamination

(Eley,1996).

25

Salmonella may be associated with all kinds of food contamination

of meat (cattle, goats, chicken, etc.) may originate from animal

salmonellosis, but most often it results from contamination of

muscles with the intestinal contents during evisceration of animals,

washing, and transportation of carcasses. When contaminated

meat is ground beef, multiplication of Salmonella may occur with

in the ground beef and if cooking is superficial, ingestion of this

highly contaminated food may produce salmonella infection

(Kenneth, 2005).

Salmonella within meat or other products may survive if cooking

temperatures and time of cooking are adequate to cook only the

surface of food, there can be no tolerance of any level of

Salmonella infection in foods ready for serving (Robert et al.,1998).

The infective dose for Salmonella infection in most commonly

given as 103 to 105 organisms but can be as high as 109 or 1010,

depending on the serotype and the host (Robert et al.,1998).

The condition needed for an outbreak is the ingestion of live cells

present in the food. Heat treatment of foods by cooking or

pasteurization is sufficient to kill Salmonella (Robert et al.,1998).

In many developing countries typhoid fever is quite common, while

it is rare in food borne morbidity (D’Aoust, 1989).

Many foods, particularly those of animal origin and those subjected

to sewage pollution, have been identified as vehicles for

transmitting these pathogens to human beings and spreading them

to processing and kitchen environments (ICMFC, 1998).

Certain groups of people, are more at risk than others for example,

infants and young children are more at risk than healthy adults

because their immune systems are not fully developed as adults;

also the elderly people are at risk because their immune systems

26

have weakened with age. People whose immune systems are not

functioning properly, such as those suffering from cancer, acquired

immune deficiency syndrome or other disease, are more likely to

get a food borne illness than healthy adults as well as pregnant

women and their fetuses (Wanger,2000).

Food poisoning is a major public health concern world wide, it

usually present as an acute enteric diseases. Thisdisease is

preventable so the knowledge of good microbiological practices

at all stages of the food production process is an essential part to

prevent food poisoning by improving food safety (Eley, 1996).

1.5. Meat Hygiene and control of pathogens: The concept of meat safety and quality assurance had evolved

with expansion in animal and meat trade. It was shown that risk

may arise from incision of lymph node during meat inspection and

consequent contamination of meat. So from cold storage, chilling

or freezing they enhance meat keeping quality (Asha,1999).

The objective of food processing and preparation is to provide safe

and nutritious food to the consumer.The responsibilities for

accomplishing this objective lie with every step in the food chain,

beginning with food production and continuing through processing,

storage, distribution, and retail sale. Consumption-processor

obligations are to process raw foods in a manner that minimizes

growth of existing microorganisms as well as avoid additional

contamination during processing (Wallace et al. 1998).

Good food hygiene practice means limiting multiplication of

contaminating organisms to as great degree as possible by

controlling the conditions under which food is processed and

stored (Eley,1996).

27

Food hygiene relates to the safety of food at all stages which lead

up to its consumption: from production through processing and

storage to the circumstances in which it is eaten, to reduce the

hazards associated with contaminated growth, which is itself

dependent on number of factors such as nutritional content of the

food, temperature, pH, gaseous conditions, presence of inhibitors,

and water activity. All these factors affect microbial activity in meat

(Eley, 1996; Judge et al., 1990).

Food may be contaminated by infected food handlers who practice

poor personal hygiene or by contact with water contaminated by

human sewage. Knowledge of basic food preparation techniques,

such as adequate refrigeration and thorough cooking of food,

should be stressed at all levels (Eley, 1996).

Improper food handling can increase food–borne illness by

allowing infectious agents to increase in numbers or by allowing for

cross-contamination between raw and cooked foods (Wallace et

al., 1998). To reduce the risk for cross-contamination, consumers

should use soap and hot water to wash hands, utensils, and other

meat products (CDC, 2004).

Food borne illnesses are important public health problems world

wide, affecting both developed as well asnon-developing countries,

leading to substantial costs in public health terms and serious

losses in terms of morbidity and mortality (Gomes et al., 1997).

Control of food safety and quality is an integral part of national

programs for development. National food control systems are

designed to protect the health and welfare of the consumer, to

promote the development of trade in food and food products, and

to protect the interests marketer against dishonest and unfair

competition (Andrews,1992).

28

As long ago as 1955, Wilson observed that it is far more important

to lay down a strict code for preparation or processing of food and

see that it is carried out properly than to rely on bacteriological

sampling of the finished product (Andrews, 1992).

Control of bacteria in meat depends on rigorous asepsis,

sterilization of equipment and disinfection (Jawetz et al., (2001).

Clean environment of food preparation is good hygienic practices

and implementation of rigorous sanitation standards in the abattoir

will help; the latter should include cleaning of items such as walls,

floors, cutting tables, knives and other utensils, operatives, clothes,

etc (Forsythe and Hayes, 1998).

Although some classic approaches to food safety rely heavily on

the end product, improving the quality of all processing steps will

result in properly safe products (Jay, 2000).

There is another way to make meat safe, although it is yet

commonly used in this country. That method is sterilization of

foods by X-ray irradiation. This treatment penetrates meat and

other foods to kill all living organisms, but dose not cook the food

(Teresa,1999).

Important measures to prevent food poisoning include educating

food workers in safe food-handling techniques and proper personal

hygiene (such as washing hands after defecation), properly

heating foods to kill pathogens and holding foods under

appropriate conditions to avoid bacterial multiplication

(ICMFS,1989).

Ground beef and other meat products should be cooked

thoroughly to internal temperatures of at least 1600 (710C), by

using meat thermometers. In the future irradiation or other

29

treatments may greatly reduce contamination of raw meat (CDC,

2004).

In control of slaughtering, the emphasis tends to be on animal

health via ante-and post-mortem inspection of animals and

carcases, because the concern has to keep meat from diseased

animals out of food channels and to ensure that slaughter and

processing operations are performed under sanitary condition

(Eely, 1996).

Hazard analysis critical control point (HACCP) is an approach to

hazard identification, assessment and control, also it is a particular

way of controlling food manufacture and handling, processing and

operating practices (Wallace et al.,1998). The goal of HACCP

system is to produce foods that are free of biological, chemical,

and physical hazards. So it is designed to prevent problems

before they occur (Forsythe and Hayes, 1998).

Consumer has a reasonable expectation that foods have been

produced and processed under hygienic condition and must have

information on both composition and nutritional aspects of products

(Wallace et al., 1998).

30

Chapter Two

Materials and Methods

2.1. Methods of sterilization: 2.1.1. Dry heat: 2.1.1.1.Red heat : By using the flame, it was used for sterilization of wire loops,

straight wires and forceps. It was done by holding the object near

and vertical to the flame until it became red (Cruickshank, R.,

Duguid, J.P., Marmion, B.P and Swain R.H, 1975).

2.1.1.2.Hot air Oven:

This method was used for sterilization of Petri-dishes, pipettes,

tubes, flasks and bottles. Materials are exposed for one hour at

1600C (Stainer, R.X,Ingraham, J.L., Weelis,M.L.and Painter,1986).

2.1.2. Moist Heat: 2.1.2.1. Autoclaving: This method was used for sterilization of culture media and

materials that could not withstand the dry heat. The exposure time

was 15 minutes at 1210C under 15 pounds pressure (Barrow and

Feltham, 1993).

2.1.3. Filtration: This method was used for sterilization of serum and potassium

tellurite solution (ISO,1999).

2.1.4. Disinfection of media preparation room:- Phenol disinfectant and 70% alcohol were used for disinfecting

floor and benches for media preparation.

31

2.2. Diluents and Solutions: 2.2.1. Buffer Field's Phosphate Buffer: 43g of potassium dihydrogen phosphate were added to 500ml

distilled water, mixed, dissolved and sterilized by autoclaving at

1210C for 15 min (Andrews,1992).

2.2.2. Physiologyical Saline: 8.5g of sodium chloride were added to 1 litre distilled water,

dissolved and sterilized by autoclaving at 1210C for 15min

(Andrews, 1992).

2.2.3.Rabbit Plasma: Blood was collected with a sterile syringe from rabbits and

centrifuged to get sterile plasma for coagulase test.

2.2.4. Potassium tellurite Solution: One gram of potassium tellurite was dissolved in 100ml water with

minimal heating, the solution was sterilized by filtration using 0.22

um pore size membranes (ISO 6888-1, 1999).

2.2.5. Iodine-Iodide Solution: Twenty-five grams of potassium iodide were dissolved in 10 ml of

water, and then twenty grams of iodine were added and diluted to

100 ml with sterile water (ISO 6579, 2002).

2.2.6. Egg Yolk Emulsion:- Fresh hen eggs were used after cleaning the eggs with a

detergent, and then immersed in 70% ethanol for 30sec.

Under aseptic conditions, eggs were broken and the yolk was

separated in a sterile container and sterile water was added about

32

four times the yolk volume, mixed and heated in water bath at

470C for 2h (ISO -1.1999).

2.2.7. Urea Solution: It consisted of 400g urea which were dissolved in 1 liter water,

sterilized by filtration and then stored in a cool place.

2.3. Cultural Media: All cultural media used for isolation and identification of bacteria

were recommended by the International Organization for

Standization (ISO).

2.3.1. Solid Media: 2.3.1.1. Baird –Parker Medium (Oxoid Code-CM275): Sixty –three grams which consisted of 10g tryptone, 5g beef

extract, 19g yeast, 10g sodium private, 12g glycine, 59g lithium

chloride and 20 g agar were suspended in 1 liter of distilled water,

boiled and sterilized. The pH was 7.0+ 0.2.

The medium was then cooled to 480C and 5mL of sterile egg yolk

and 1 ml potassium tellurite were added to 95 ml melted base and

mixed before pouring 15 ml volumes into sterile petridishes.

2.3.1.2. Xylose Lysine Deoxycholate (Mast code-DM 23OD): Consisted of 7.5g lactose,7.5g sucrose, 5g L-lysine, 5g Nacl, 3g

yeast extract, 3.7g xylose, 2.5g dexoycholate, 6.8g sodium

thiosulafate, 0.8 ferric ammonium citrate, 0.08 g phenol red and

15g agar . About fifty-six grams were dissolved in 1 liter distilled

water, boiled and cooled to 500C, then poured as 15ml volumes

into 65 plates. The pH was 7.4 + 0.2.

33

2.3.1.3. Brilliant green agar: This medium consisted of 18.6g ox-bile (purified) 7g peptone, 7g

lactose, 0.013 brilliant green and 15g agar. Fifty-six grams were

suspended in 1 litre of distilled water, boiled and cooled to 500C,

then poured into plates in volumes of 15ml. The pH was7.4.

2.3.1.4.Nutrient agar (Oxoid code-CM3):

Twenty three grams which consisted of 3g meat extract, 5g

peptone and 15g agar were suspended in 1 liter of distilled water,

boiled and sterilized by autoclaving at 1210C for 15min.The

medium was poured into sterile petridishe in 15ml volumes. The

pH was 7.4.

2.3.1.5. Eosin methylene blue agar (Mast code-DM133D): It consisted of 10g of each peptone and lactose, 2g of K2

HPO4,0.4g eosin-y, 0.06 methylene blue and 15g agar. Of the

medium 37.5g were dissolved in 1 litre distilled water, boiled and

sterilized by autoclaving then poured into 35 sterile petridishes.

The pH was 7.1+ 0.2.

2.3.1.6. Urea agar base (Oxoid Code-CM 0053): This medium consisted of 1 g peptone, 1 g glucose, 5g sodium

chloride, 2g potassium dihydrogen phosphate, 0.01 phenol red and

15g agar. 24grams were suspended in 95 ml of distilled water by

heating. The pH was 7.4.The medium was sterilized by autoclaving

at 1210C for 15 min. It was then cooled to 500C and sterile 40%

urea (5ml) were added. After mixing well, it was distributed as 10

ml into sterile bottles and set in a slope position.

34

2.3.1.7. Triple sugar iron Agar (Oxoid code –CM 227): It consisted of 20 g peptone, 10g lactose, 10g sucrose, 5g sodium

chloride, 3g meat extract, 3g yeast extract, 0.02 phenol red and 15

g agar.68 grams were dissolved in water by heating, then sterilized

by autoclaving at 1210C for 15min. The pH was 7.4. Then

dispensed into test tubes (10ml) and solidified in the slope position.

2.3.2. Liquid Media: 2.5.2.1. Buffered peptone water (Mast code-DM 494D):

The medium consisted of 10 g enzymatic digest of casein, 5 g

sodium chloride, 9g (Na2HPO4.12 H2O) and 15g (KH2 PO4). Of the

medium 25.5 grams were dissolved in water by heating and

sterilized by autoclaving at 1210C for 15 min. The pH was 7,

before sterilization. The medium was then distributed as 225 ml

into suitable containers.

2.3.2.1. EC medium (Difco code- 135581 XA): This medium consisted of 20 g tryptose, 5 g lactose, 4 g (K2H

PO4), 1.5g bile salts and 5g sodium chloride. Thirty grams were

suspended in 1 liter distilled water and dissolved. The pH was 6.9,

then distributed as 8ml into test tubes containing inverted

fermentation vials. Then sterilized by autoclaving for 15 min at

1210C.

2.3.2.2. Selenite cystine (Oxoid code-CM 699): Consisted of 5g tryptone, 4g lactose, 4g sodium selenite, 10g (Na2

HPO4) and 0.01g cystine. Of the medium 22g were suspended in 1

liter of distilled water and boiled. The pH was 7. It was then

distributed in 10ml volumes in test tubes. The medium was heated

for 10 min in steamer before being used.

35

2.3.2.3. Tetrathionate Base Broth (Mast code-DM 2195): The medium consisted of 5g polypeptone, 10g calcium carbonate,

30g sodium thiosulfate 5 H2O and 1g bile salts. Forty –six grams

were suspended in 1 litre distilled water, mixed, and boiled and

sterilized by heating. The medium was cooled to 450C then stored

at 5-80C. The pH was 8.4.

2.3.2.4. Barin heart Infusion (Oxoid code-CM 225): This medium consisted of 12.5 g calf brain in fusion, 10 g peptone,

5g beef heart infusion, 2g glucose, 2.5g (Na2 HPO4) and 5g

sodium chloride. Thirty seven grams were dissolved in 1 liter

distilled water, then dissolved and distributed 0.5ml into small test

tubes and autoclaved at 1210C for 15 min.

2.3.2.5.Lauryle sulfate tryptose broth (Oxoid code-CM0451): This medium consisted of 20g tryptose,5g lactose, 2.8g (KH2PO4)

and 0.1g sodium lauryle sulfate. Twenty eight grams were

suspended in 1litre distilled water, the pH was 6.8 then dispensed

into suitable tubes containing inverted fermentation tubes and

autoclaved at 1210C for 15min.

2.3.2.6. Peptone water (Oxoid code-CM9R4): Fifteen grams which consisted of 10g peptone and 5g Nacl were

added to 1 liter of distilled water, mixed, distributed into tubes after

adjusting the pH to 7.4 and sterilized by autoclaving at 1210C for

15min.

2.3.2.7. Methyl red and Voges Prokauer media (Oxoid code-CM 43R 26):

It consisted of 5g peptone, 5g dipotassium hydrogen phosphate

and 1000 ml distilled water, which were mixed, steamed to

dissolve and filtered.

36

The pH was 7.5 and the medium was sterilized by autoclaving at

1210C for 10 min then distributed as 5ml into sterile tubes.

2.4. Reagents: 2.4.1. Kovac’s reagent: It consisted of 5g 4-dimethylamino-benzaldehyde, 75ml of amyl

alcohol and 25ml concentrated hydrochloric acid. The aldehyde

was dissolved in the alcohol and added to HCL slowly. The

reagent was protected from light and stored at 40C.

2.4. Voges- Proskauer test reagent: It consisted of two solutions:

(1) Alpha-naphthol solution consisted of 5% alpha-naphthol in

ethanol. (2) 40% of potassium hydroxide solution.

2.5. Sampling: The subject of the investigation was processed meat. Samples

were collected from the final product of sausage, minced meat and

burger.

2.5.1. Description of sample: 2.5.1.1. Definition of meat: Meat is defined as those animal tissues, which are suitable for use

as food. All processed or manufactured products, which might be

prepared from those tissues, are included in this definition (Judge

et al., 1990). Also meat is defined as the flesh of animals used as

food, and it is often widened to include, as well as the musculature,

organs such as liver and kidney, brains and other edible tissues

(Lawrie, 1990).

37

2.5.1.2. Minced Meat: Beef is freed from bone and fascia, and then minced by using

electrical mincing machine. The preparation of minced meat

involves fragmentation of meat tissue with liberation of meat juices

and therefore bacteria normally present on the meat surface are

spread throughout the entire product.

2.5.1.3. Sausage: Sausage consists of mined meat to which spices and sodium

chloride were added and intestine or gelatinous casings were

used. The mucus membrane of the intestine was carefully

removed using a knife, and then washed by passing water through

it. The minced meat is mixed with pepper, parsley and garlic, and

then pushed inside the intestine or gelatinous casing with special

instrument to form finger-like shape of 5.7 cm in length.

In factories special machines are used to produce large quantities.

2.5.1.4. Beef burger: Beef burger consists of minced meat, pepper and sodium chloride.

The mixture is pressed lightly on a metal object of 8–10cm in

diameter and 0.6-1.0cm in thickness.

2.5.2. Sources of samples: Samples were collected from six meat processing factories in

Khartoum state located in Khartoum, North Khartoum, and

Omdurman.

2.5.3. Collection of samples:

A total of 75 frozen samples; 25 from each meat type were

collected randomly during period eleven months from April 2007 to

February 2008.

38

The weight of each unit were 500 gram distributed in plastic bags

by the producers.

2.5.4. Transportation of collected samples: The samples were transferred to the laboratory in thermos flasks

containing ice, as quick as possible to avoid contamination of

samples (Androw, 1992).

2.5.5. Preparation of the samples: In the laboratory samples were thawed in a refrigerator (40C) for

18hr.To isolate E. coli and coagulase positive staphylococci, 50g

of each representative sample were aseptically weighted in sterile

bag mixed with 450ml buffer phosphate and blended to get

homogenous initial suspension using stomacher blender.

To isolate Salmonella; 25g of each representative sample were

aseptically weight and blended with 225ml buffered peptone

water to get homogenous suspension.

2.6. Cultural Methods: All methods of isolation and identification of bacteria under study

were carried out according to the International Organization for

Standardization (ISO).

2.6.1. Method for Presumptive E.coli Detection: For detection of E.coli, the homogeneous samples were inoculated

in selective enrichment lauryle sulfate tryptose broth, 10ml of

homogeneous samples were added to 50ml lauryle broth and

incubated at 370C for 24-28h. Then broth culture with positive gas

tubes was inoculated on selective Ec medium in tube, containing

Duham’s tubes and incubated at 450C for 24-48h.

39

EC cultures which produced gas in Durham’s tubes were

considered positive tests for presumptive E.coli. These were

confirmed by streaking on eosin-methylene blue agar which was

incubated at 370C for 24h. The typical colonies of E. coli appeared

as a dark centered with or without green metalic sheen. Isolated

colonies were confirmed by biochemical test after subculture in

nutrient agar to get pure cultures (ISO 7251, 1993).

2.6.2. Method for Salmonella isolation and Identification: For isolation of Salmonella, samples were enriched in non-

selective medium of buffered peptone water for 24 hr. One ml of

each sample was added to 10ml selenite cystine and also to 10 ml

of tetrathionate broth and in cubated at 370C for 24hr. Growth from

each medium was inoculated on xylose lysine deoxychcholate

agar and brilliant green phenol red agar which were incubated at

370C for 24h.The typical colonies of Salmonellae were pink with or

without black centers or black colonies. A few Salmonella species

produce a yellow colonies. Suspected colonies were picked five

times from each selective agar. Isolated colonies were confirmed

when used appropriate biochemical test (TSI agar, urea agar, VP

test, indole test) and serological tests by using antisera after sub

culture on nutrient agar(ISO 6579, 2002).

2.6.3. Method for isolation and enumeration of staph.aureus: For isolation of staph. aureus bacteria, the homogeneous samples

were inoculated on a solid selective Baird-Parker medium.

Aseptically transfered 1ml inoculums of the initial suspension

(diluted1/10) was spread over the surface three agar plates.

The plates incubated aerobically at 370C for 24-48h. The typical

colonies were moist black or grey, shining and convex and were

40

surrounded by a clear zone. For enumeration each colony type

appeared as S.aureus was counted.Only plate that contained 30-

300 was selected. More than 1 colony of each type counted and

tested for coagulase production. Isolated colonies were confirmed

when inoculated into a small tubes of brain-heart infusion and

incubated at 370C for 24h. Aseptically 0.5mL plasma was added to

each culture and mixed, the cultures were incubated at 370C and

examined periodically over 6hr period for clot. Colt which found in

tube when inverted was considered a positive reaction. Number of

colonies on set of triplicate plates which represented by colonies

giving positive coagulase test added and multiplied by sample

dilution factor and reported this value as number of S.aureus/g of

product tested (ISO 6888-1, 1999).

2.6.4. General Examination:

General morphology, color, shape and size of colonies, were

examined with the naked eye. Sugars fermentation indifferent test

was observed and recorded. Positive coagulase reaction and the

positive reaction which indicated by development of colors was

observed and recorded.

2.6.5. Primary testes:

2.6.5.1. Gram staining & Microscopic Examination: Gram stain was used to study morphology, shape and gram

staining reaction of each isolate. A sterile loop was used to prepare

a suspension from a single colony in normal saline on a clean

slide.

In microscopic clean slides smears were made from isolates and

fixed with heat. Crystal violet stain was powered on the slides for

41

one minute and washed. The slides were then covered with Lugols

iodine for 1 min and washed off with water, decolorized by 70%

alcohol for 30 seconds and rinsed with water. Carbol fuchsion was

added for one minute and the slides were examined under the oil

immersion Lens (100X).

Gram-positive bacteria were blue and Gram negative bacteria

were red. Shapes and arrangements of organisms were identified

according to Barrow and Feltham (1993).

2.6.5.2. Sugar fermentation tests: The lauryle sulfate broth was inoculated with the test culture,

incubated at 370C for 48hr. Gas production developed an empty

space in Durham’s tube.

2.6.6. Secondary tests: 2.6.6.1. Urease activity test: The pure cultures of selected colonies were incubated in slant

surface medium by sterile loop, and incubated at 370C for 24h.

Change in color to pink indicated a positive reaction.

2.6.6.2. Triple sugar iron (TSI) test: The tested bacteria were streaked on the agar slant surface and

stabbled in the butt and incubated at 370C for 24h. Change in color

to yellow and black in butt (formation of hydrogen sulfide) and red

in slant that indicated a positive reaction for salmonella. And then get yellow in both slant and butt with formation of gas at

bottom that indicated a positive reaction for E.coli .

2.6.6.3. Voges – Proskauer (v.p) test: Glucose phosphate medium was inoculated with each test culture

and incubated at 370C for 48h, then 0.6 ml of 5% alpha–naphthol

42

solution and 0.2ml of 40%KOH solution were added to 1ml of

culture media, and shaken well. The tubes were stoped and

examined after 15min and 1h. Strong red color indicated a positive

reaction.

2.6.6.4. Indole test: The test culture was inoculated in peptone water medium and

incubated 370C for 24h. One ml of kovac’s reagent was run down

the tube; the positive reaction gave a pink ring within minutes.

2.6.6.5. Tube coagulase test: 0.5ml of brain-heart infusion was inoculated with the test culture,

and incubated at 370C for 24h, 0.5 ml of rabbit plasma was added

and incubated again at 370C for 2- 6 hrs. Tubes were examined

after 4hr to 6 hr for clotting. If the volume of clot occupied more

than half of the original volume this was regarded as a positive

reaction. Negative tubes were re-examined after 24hr of incubation

at 370C for coagulation.

2.6.7. Slide agglutination test for Salmonella: This test was done by placing one drop of saline solution into a

clean glass slide. The tested colony was mixed to get a

homogeneous suspension. Then a drop of the appropriate sera (O-

polyvalent anti serum A-S) for salmonella was added .The whole

suspension was mixed well for 30s to 60s and if agglutination

occurred, the reaction is considered positive.

43

Chapter Three

Results

A total of seventy five samples obtained from different meat

factories distributed in Khartoum State, for processing many kinds

of meat products, were examined for some pathogenic bacteria

(Salmonella, Staphylococcus.aureus and pathogenic Escherichia

coli).

Twenty-five samples were examined from each frozen minced

meat, frozen sausages and frozen beef burger.

A total of 77 isolates of the three types of organisms were obtained

from the different products (table 4).

3.1. Prevalence of different species: Staphylococcus.aureus was the most frequent organism, which

was found in 40 samples, with a prevalence of (53.3%) (table 3)

followed by E. coli which was found in 21 samples with a

prevalence of (28%) (table 1) then Salmonella which was found in

16 samples with a prevalence of (21% ) (table 2).

3.1.1. E. coli: The positive samples which were identified in minced meat were 6

with a prevalence rate of (24%) (table.1). In burger the positive

samples were 10, with a prevalence rate of (40%) (table.1). In

sausage the positive samples were 5, with a prevalence rate of

(20%) (table.1 & figure.1).

44

3.1.2. Salmonella: Salmonella was indentified in 7 samples of minced meat (28%)

and in 5 samples of sausage (20%) where as only 4 samples of

burger (16%) were positive (table 2 & figure.2).

3.1.3. Staph. aureus: Staph.aureus was found in 13 samples (52%) of minced meat, in

9 samples (36%) of burger and in 18 samples (72%) of sausage,

were positive (table 3 & figure.3).

45

Table (1): Shows the prevalence rates of E.coli in the different meat products:

Table (2): Shows the prevalence rates of Salmonella in the different meat products:

Meat product No. of samples No. of the positive samples

Prevalence rate

Minced meat 25 7 28%

Burger 25 4 16%

Sausage 25 5 20%

Total 75 16 21%

Meat product No.of samples No. of the positive samples

Prevalence rate

Minced meat 25 6 24%

Burger 25 10 40%

Sausage 25 5 20%

Total 75 21 28%

46

Table (3): Shows the prevalence rates of staph.aureus in the different meat products:

Meat product No. of samples No. of the positive samples

Prevalence rate

Minced meat 25 13 52%

Burger 25 9 36%

Sausage 25 18 72%

Total 75 40 53.3%

Table (4): Shows comparison between the no. of different bacterial isolates from different meat products.

Meat product No. of total types of Isolates

Minced meat 26

Burger 23

Sausage 28

47

Figure (1): Shows the prevalence rates of E.coli in different meat products:

48

Figure (2): Shows the prevalence rates of Salmonella in different meat products:

49

Figure (3): Shows the prevalence rates of staph.aureus in different meat products:

50

Chapter Four Discussion

Meat surface is usually heavily contaminated with a wide range of

microorganisms and due to its high nutritional value (water,

proteins, peptides, aminoacids, nucleotides, sugars, minerals and

vitamins) it is a suitable medium for the development of most

bacteria (Steinhauser,1995).

The microbial counts on the final products depend on the initial

count in carasses, ambient temperatures involved, personal

hygiene, efficiency of the sanitary program applied, changes in the

water activity of the meat surface and the general management

applied throughout the meat production (Nortije, G.L, Nel, L.,

Jordan, E., Badenhorst K., Geodhart, E. and Holzapfel,

W.H,1990).

One of the most important methods for quality control is using

microbial standards with microbiological examinations. These

examinations include total bacterial count, coliform count and

survey of potentially pathogenic or pathogenic bacteria including E.

coil, S.aureus, Salmonella spp, Listeria moncytogenes, and

Cl.perfringens ( Hamid and Majid,2008).

In this study E.coli, Salmonella and S.aureus were isolated and

identified from processed beef samples (minced meat, Burger and

sausage). The bacterial counts were found to be above the

acceptable range of Sudanese microbiological limits for processed

meat products which aimed to protect the consumer by providing

healthy and safe food.

51

Dawdell and Board (1980) reported that meat products like

sausages, beef-burgers and similar meat products usually contain

cereals or rusk, salt and spices in addition to minced meat. These

additives increase the general viable count which may include

some dangerous pathogenic and spoilage bacteria, as usually

there is no previous treatment of spices to reduce their microbial

content. Spices and other additives were found to be a high source

of contamination (Amanie, 2000). This point was also emphasized

by Frazier and Westhoff, (1988).

Fatima (1990) found that Freezing at -200C injured bacterial cells.

Lyla (1987) found that at -200C the death of E.coli and S.aureus

was proportional to the storage time. Samples used in this study

were kept at-200C and despite injury from freezing all pathogenic

bacteria under study were detected in most frozen samples.

A high prevalence rate of pathogens under study were recorded

from the three types of meat product; 77 isolates from 75 sample.

S.aureus had a (53%) prevalence rate followed by E.coli (28%)

and Salmonella (21%). Sausage was the most contaminated type

of meat product followed by minced meat then burger as each type

was treated and prepared in a different way under unsanitary

conditions, also contamination may have originated before mincing

during slaughter and skinning. Further more, the addition of

untreated spices contributes to contamination.

In this study bacterial examination of processed beef samples was

made by conventional bacteriological methods, the use of more

sensitive methods like polymerase chain reaction (PCR) possibly

would have increased the detection rate of different bacteria.

The PCR assay has been used to identify Salmonella in food and

clinical samples (Cohen, N.d, Neibergs, H.L, and Hargis, 1993).

52

Narasimha and Ramesh (1988) found that the bacterial growth in

minced meat was dependent on three main factors. They include

the bacteriological quality of the meat used for mincing, the

cleanliness of equipment and the time and temperature of storage.

Escherichia coil is an enterobacterium, and it is presence in large

number in food indicates feceal contamination and a possible

hazard of infection with enteric pathogens (Harrigan and Margaret,

1976; Omer, 1990; Bolton, 1996). In this study E.coli was most

next predominant in products possibly due to fecal contamination

and the addition of un cooked ingredients, accompanied with poor

sanitation during processing. Similar results were made by

Mohamed (1991), Elhussien (2003) and Wafa (2004).

E.coli is considered one of potentially pathogenic related to meat

(Federal Register U.S.A,1999; food safety and inspection Services,

1997). The total prevalence rate of E.coli in all samples under

study was 28% this agrees with Vorstar et al (1994) who recorded

a prevalence rate of E.coli in (27%) of his processed meats. In this

study beef burger had recorded a highest percentage of E.coli

(40%) out of 25 samples. This may be due to initial contamination

of ground meat with bacteria as (24%) had E.coli. However

El.Gohary (1994) found a higher prevalence rate (75%) of minced

meat samples had E.coli .

Wigner (2000) and cheesbrough (2000) reported that meat and

meat products were most frequently contaminated with Salmonella

spp. Hugas et al (1995) and kleemann and Bergann (1996)

isolated Salmonella from raw sausage. In this study Salmonella

was isolated from all frozen types of meat product samples under

study, this result agreed with Fatima (1982) and Wafa (2004), but

53

disagreed with the finding of Hussein (1975) and Amanie (2000),

who failure to isolate it from their frozen samples.

Salmonella recorded high rate of isolates in minced meat product

(28%) compared with sausage (20%) and beef burger (16%) this

disagreed with Wafa (2004) who failured to isolate it from her

frozen samples, and nearly to result made by Edel and

Kampelmacher (1971) who found that (33.7%) of minced meat

samples, (16%) of beef burgers and (16.9%) of raw sausages were

contaminated with Salmonella.

Roberts et al (1975) found that, the prevalence of Salmonella in

sausages from several different producers to range from (2-60%)

such contaminated meat and meat products may either cause

direct infection or indirect contamination through cross-

contamination in kitchen.

The use of prerichment technique which was employed in this

study probably would have increased the recovery rate. In spite of

that Salmonella recovery has a lower percentage rate (21%) out of

all total samples under study compared with E.coli (28%) and

S.aureus (53%) and that may be as a result of their sensitivity to

injury due to freezing, also according to their prevalence in

environment .

Staph. aureus was found in beef products by many investigators,

it was detected in meat samples by Wyatt and Guny (1980) and

Duitschaever et al. (1977), Jay (1986) reported that coagulase

positive S.aureus was isolated from raw beef fried, beef burger and

sausage.

Since (50-60%) of the normal human population carry coagulase–

positive staphylococci on their skin and nasopharynx (Angelotti,

1970) and in their arms and hands as reported by (Banwart,

54

1981).The isolation of S.aureus from meat products with a

prevalence rate of (53%) in this study indicates that carriers were

involved in contaminating meat processing especially the

processing stage was done under poor hygienic conditions.

Meat handlers in Sudan are not subjected to medical check and

may constitute a public health hazard. These workers may be not

receive any hygienic health education training courses.

Staphylococcus aureus may constitute a public health hazard

since small number of the organism can proliferate during handling

of meat. The increase of the growth rate leads to large bacterial

population which may result in accumulation of toxic metabolites

and subsequent food poisoning (Samia, 1997).

Generally, in the present work S. aureus was isolated from all

types of product ; this agrees with the findings of Mohamed (1991)

and Mohamed (1997) who isolated it from most of their samples

while Wafa (2004) was not able to recover it from her frozen

samples.

S.aureus was isolated from a large number of sausage and minced

meat samples, (72%) and (52%) respectively. This is a higher

percentage compaired with result made by Samia (1997) who

recorded a prevalence rate of (43%) and (15%), where as Amanie

(2000) and Hussein (1975).

Sausage has a high percentage of recovery (72%) out of 25

samples of S.aureus. This high prevalence rate may be attributed

to many factors: inadequate washing of intestines before

processing, might directly increase the rate of Staphylococci, the

main component of sausage is ground meat and it was initially

found to record a high percentage rate (52%) of S.aureus, and the

55

addition of spices and sodium chloride to the sausage during

processing and the time which

needed to give the special taste for it; all these factors give a

suitable environment for the growth of Staph.aureus, finally

handing and keeping sausage frozen for a long time also may

facilitate the growth of this organism.

56

Conclusion:

This study revealed that there was a high level of contamination of

processed meat with pathogenic food poisoning bacteria which can

reduces the quality of meat products and make proper working a

necessary

The detection of potentially pathogenic or pathogenic organisms

like E.coli, Salmonella and Staph.aureus with appreciable

percentage constitutes a public health hazard.

Recommendations:

1. Control of entero pathogenic E.coli and other food-borne

pathogens such as Salmonella and Staphylococcus aureus

can be achieved. Precautions should include adequate

cooking and avoidance of cross contamination of cooked

meat by contaminated equipment, water or infected food

handlers.

2. Food service establishments should monitor adequacy of

cooking holding times, and temperature as well as personal

hygiene of food handles with good health education courses,

for those workers in such meat factories.

3. Since meat is a highly perishable food and is subject to

several changes, every effort should be made during

handling and processing to make it reach the consumer in an

acceptable and safe form.

57

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