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The Islamic University Gaza Deanship of Graduate Studies Faculty of Science Biological Sciences Master Program Medical Technology Antimicrobial Resistance for Enteric Pathogens Isolated from Acute Gastroenteritis Patients in Gaza strip, Palestine. By Nahed Abdelateef Supervisor Dr. Abdelraouf A. Elmanama Ph. D Microbiology A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Biological Sciences/ Medical Technology May 2011

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Page 1: Antimicrobial Resistance for Enteric Pathogens …library.iugaza.edu.ps/thesis/95584.pdf · Antimicrobial Resistance for Enteric Pathogens Isolated from Acute Gastroenteritis Patients

The Islamic University – Gaza Deanship of Graduate Studies Faculty of Science Biological Sciences Master Program Medical Technology

Antimicrobial Resistance for Enteric Pathogens Isolated from Acute Gastroenteritis Patients in Gaza strip, Palestine.

By

Nahed Abdelateef

Supervisor

Dr. Abdelraouf A. Elmanama

Ph. D Microbiology

A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Biological Sciences/ Medical Technology

May 2011

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Declaration "I herby declare that this submission is my own work and that, to the best of

my knowledge and belief, it contains no material previously published or

written by another person nor material which to a substational extent has

been accepted for the award of any other degree of the university or other

institute, except where due acknowledgment has been made in the text".

Nahed Abdelateef

© Copyright by

Nahed Abdelateef

2011 Copyright. All Right Reserved: No part of this work can be copied, translated or stored in retrieval system, without prior permission of the author.

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DEDICATION This thesis is dedicated to my family and

to my friends who supported me all the

way since the beginning of this study.

This thesis is also dedicated to all those

believe in the richness of learning.

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List of contents Declaration ……………………………………………………….……….…….......... iv

Dedication ……………………………………………………………......................... v

List of figures………………………………………………………………..………... ix

List of tables ……………………………………………………………….................. x

List of abbreviations……………………………………………………….…..……… xi

Acknowledgments ……………………………………………………………............. xiii

CHAPTER I: INTRODUCTION

1.1 Overview ……………………….………..…………………………..................... 3

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

1.3 Significance ………………………………………………………………............ 5

CHAPTER II: LITERATURE REVIEW

2.1Acutegastroenteritis………………………………………………………..……….. 7

2.2 Antimicrobial resistance …………………………………………........................... 7

2.3 Diarrheal disease is the most common cause of illness ………………………...… 8

2.4 Common bacterial causes of diarrhea ……………...…………............................... 9

2.4.1 Salmonella spp history……………………….…………………….......…........... 9

2.4.2 Shigella history................................................................................…….……..... 10

2.4.3 Campylobacter history …………….…………...……………………..........….... 12

2.4.4 Yersinia enterocolitica history .………………….………………..............…..... 15

2.4.5 Aeromonas hydrophila history..………………………………….…................… 17

2.5 Epidemiology and antimicrobial resistance of gastroenteritis pathogen……..……. 19

CHAPTER III: MATERIALS AND METHODS

3.1 Materials ………………………………………………………………................. 28

3.1.1 Bacterial culture media…….………………………………………….…..... 28

3.1.2 Reagents ………………………………………………………...…….......... 28

3.1.3 Supplies ……………………………………………………..…………........ 29

3.1.4 Apparatus and equipments ………………………………….…………….... 29

3.2 Study area ……………………………………………………………………..….. 30

3.2.1 Samples …………………………………………………………………….. 30

3.2.2 Samples transport.………………………………………………...………… 30

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3.2.3 Questionnaires………………………………………………………………. 30

3.2.4 Permissions and ethical consideration ………………………...…………… 31

3.2.5 Data analysis …………………………………………………..…………… 31

3.3 Isolation and identification procedures of enteropathogenic bacteria………..…... 31

3.3.1 Salmonella and Shigella………………………………………..…...…….… 31

3.3.2 Campylobacter. …………………………………………………...…….….. 33

3.3.3 Yersinia enterocolitica ………………………………………...…………… 33

3.3.4 Aeromonas hydrophila ………………………………………...………..….. 33

3.4 Antimicrobial susceptibility for the bacterial isolates…………….…...………….. 34

3.5 Staining and biochemical tests……………………………………...………… ..... 34

3.5.1 Gram stain …………………………………………………………...…....... 34

3.5.2 Oxidase test …………………...……………………………………............. 35

3.5.3 Catalase test…………………………………………………..….................. 35

3.5.4 API 20 E test (BioMerieux, France)…………………………...…................ 35

3.5.5 API campy test……………………………………………………...………. 35

3.6 Specific antisera………………………………………………............................... 36

3.6.1 Serotyping of Shigella spp……………………………….............................. 36

3.6.2 Serotyping of Salmonella spp……………………......................................... 36

CHAPTER IV: RESULTS

4.1 Description of study samples…………...………………………………………… 37

4.1.1 Distribution of the samples in terms of residence area of the study samples.. 37

4.1.2 Sex and age distribution of the study samples ……………...………..…...... 38

4.1.3 Socioeconomic status of the study samples ……………………...……….... 38

4.2 Isolation and identification of enteropathogenic bacteria……………...…..…....... 39

4.2.1 Salmonella and Shigella…………………………………............................... 39

4.2.2 Campylobacter ................................................................................................ 40

4.2.3 Yersinia enterocolitica ………………………………………...……………. 41

4.2.4 Aeromonas hydrophila ………………………………………...………......... 42

4.2.5 Number and types of isolated enteropathogenic bacteria among population.. 43

4.2.6 Isolated enteropathogenic bacteria distributed by residence…….…………... 43

4.2.7 Occurrence of enteropathogenic bacteria among the different age groups….. 44

4.2.8 Distribution of enteropathogenic bacteria according to gender……..…...….. 45

4.2.9 Clinical symptoms in relation to presence of isolated bacteria…………....… 46

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4.2.10 Correlation of the socioeconomic status of the study samples with isolated

enteropathogens………………………………………………..…...……… 47

4.3 Antimicrobial susceptibility profile…………………………..………………....... 48

4.3.1 Antimicrobial profile of enteropathogenic bacteria isolated from patient...... 48

4.3.2 Prescription of antibiotics to the diarrheal patients…………….…................ 50

4.4 Correlation of socioeconomic factors with diarrhea………………….…...……... 51

4.4.1 Correlation between diarrhea and domestic animals in the houses….....…... 51

4.4.2 Correlation between diarrhea and some furniture in the house…….........…. 52

4.4.3 Correlation between diarrhea and current water access in the houses…….... 52

4.4.4 Correlation between diarrhea and drinking water in the house…....……...... 53

4.4.5 Correlation between diarrhea and sewage disposal…………...…………..... 54

4.5 Clinical signs and symptoms of diarrheal patients……………….…...…...……... 55

4.6 Management and follow-up of diarrheal patients ……………….….......……...… 56

CHAPTER V: DISCUSSION

5.1 Children age less than 5 years old are more susceptible to infection…………….. 58

5.2 Diarrhea and enteropathogenic bacteria is more frequent in crowded houses with

low room's number………………………………………………………………... 59

5.3 Diarrhea is more frequent in houses rearing poultry, sheeps, and pigeons……..... 59

5.4 Improvement in sewage disposal is vital in the reduction of diarrheal diseases..... 60

5.5 Correlation between water access, drinking water and diarrhea……….…..…...... 61

5.6 Sign and symptoms of diarrheal patients…………..………………….…….......... 63

5.7 Follow-up and convalescence of diarrheal patients………………………...…...... 64

5.8 Prevalence of bacterial enteropathogens……………………………….…...…...... 65

5.9 Distribution of isolated enteropathogenic bacteria by residence……….…...……. 65

5.10 Antimicrobial resistance for enteropathogenic bacteria……………....………… 67

5.11 Dehydration caused by diarrhea………………….…………………..…………. 68

5.12 Relation of the weather with isolation rate of enteropathogenic bacteria….......... 70

CHAPTER VI: CONCLUSIONS AND RECOMMENDATIONS

6.1 Conclusions ……………………………………………………………………….. 71

6.2 Recommendations………………………………………….…………………...…. 73

REFERENCES……………………………………………………….………....…..... 75

ANNEXES…………………………………………………………………..….……... 100

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List of figures

Figure (1.2): Distribution of causes of death among children……...…………. 8 Figure (1.3): Isolation and identification procedures of enteropathogenic bacteria………………………………………………………….. 32 Figure (4.1): Colony morphology for Salmonella…………………………….. 39 Figure (4.2): API 20E results for isolated bacteria……………………..……... 40 Figure (4.3): Agglutination with specific antisera for Salmonella ……............ 40 . Figure (4.4): Colony morphology for Campylobacter spp…………...…....….. 40 Figure (4.5): (Modified CCDA) incubated in microerophilic condition at 42°C…………………………………………………………... 41 Figure (4.6): Identification of Campylobacter spp. by API campy test………. 41 Figure (4.7): Colony morphology for Yersinia enterocolitica on Yersinia selective agar……………………………………………………. 41 Figure (4.8): Identification of Yersinia enterocolitica by API 20E………..… 42 Figure (4.9): Colony morphology for Aeromonas hydrophila…………...….... 42 Figure (4.10): Identification of A. hydrophila by API 20E…………................ 42 Figure (4.11): Enteropathogenic bacteria isolated from the samples……......... 43 Figure (5.1): Levels of dehydration in children with acute diarrhea…….......... 69

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List of table

Table (4.1): Primary health care centres included in this study………............. 34 Table (4.2): Distribution of the cases and controls due to sex and age……..... 38 Table (4.3): Distribution of the cases and controls due to Socioeconomic status……………………………………………………………... 39 Table (4.4): The isolated enteropathogenic bacteria by residence……………. 44

Table (4.5): Enteropathogenic bacteria among the different age group............ 45 Table (4.6): Enteropathogenic bacteria among the gender…………................ 45 Table (4.7): Clinical symptoms in relation to presence of enteropathogens..... 46

Table (4.8): Socioeconomic status of the study samples with isolated enteropathogenic bacteria…........................................................... 47 Table (4.9): Antimicrobial susceptibility of isolated enteropathogenic bacteria…………………………………………………………... 49 Table (4.10): Prescription of antibiotics to the diarrheal patients…….............. 50 Table (4.11): Domestic animals in the houses and diarrhea….......................... 51 Table (4.12): Correlation between diarrhea and some furniture in the house... 52 Table (4.13): Current water access in the houses….......................................... 53 Table (4.14): Drinking water in the houses……............................................... 54 Table (4.15): Sewage disposal and diarrhea……………………................... 55 Table (4.16): Clinical signs and symptoms of diarrheal patients…….............. 56 Table (4.17): Management and follow-up of diarrheal patients……............. 57 Table (5.1): Clinical features of infection with selected diarrheal pathogens... 64

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List of abbreviations AgaKhan University AKU Analytical profile index for campylobacter API Campy Analytical profile index 20 tests for Enterobacteriaceae API-20E Campylobacter Blood-Free Selective Agar Base CCDA Clinical and Laboratory Standards Institute CLSI Carbon dioxide CO2 Diffuse adhering Escherichia coli DAEC Deoxyribonucleic Acid DNA Enteroinvasive Escherichia coli EIEC Enteropathogenic Escherichia coli EPEC Extended spectrum beta-lactamase ESBL Enterotoxigenic Escherichia coli ETEC European Union EU Food and Agriculture Organization FAO Gastrointestinal tract GI Gram G Hektoen Enteric agar HE Hydrogen peroxide H2O2 Hydrogen sulphide H2S Flagella antigens H-antigens The International Centre for Diarrheal Disease Research ICDDR Kilometre Km Lipopolysaccharide LPS Minimum inhibitory concentration MIC Ministry of Health MOH Milli litter ML Micro gram µg Nitrogen N2 Sodium chloride NaCl Non typhoidal Salmonella NTS Somatic antigen O-antigens Oxygen O2 Oral rehydration salts ORS Oral rehydration therapy ORT Phosphate buffer saline PBS Polymerase chain reaction PCR Potential of hydrogen pH Primary health care centre PHCC Polymorphonuclear leucocytes PMNs Plasmid for Yersinia virulence PYV Surface layer S-layers Shiga toxin-producing Escherichia coli STEC United Nations UN United Nations Relief and Works Agency UNRWA United State of America USA

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Verocytotoxin-producing Escherichia coli VTEC World Health Organization WHO Wastewater treatment plant WWTP Xylose Lysine Deoxycolate agar XLD Yersinia enterocolitica biotype (BT) 1ª YE BT 1A

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Acknowledgments

I would like to express my gratitude to all people who, have contributed to this work.

In particular, I would like to thank:

Special thanks to my supervisor Dr. Abedelraouf Elmanama, for his professionally,

encouragement and enthusiastic, for his great knowledge and tremendous

intelligence. He is a true researcher driven by curiosity and with never ending energy.

Without his stimulating, critical discussions, comments, and great help, this work

would not have been completed.

Doctors of the World (Médecins du Monde - France), Gaza, Palestine, and the staff

especially Ms. Heba Elsharif, and Dr. Ahmad abuteir for their excellent help and

funding of this work.

Dr. Adnan Al-Hindi, for being such a nice and supportive person and for his, very

nice comments, discussions, and great support, and for helping me in statistical

analysis.

Dr. Randa Elkhodary, always support me, pragmatic suggestions that had a

significant influence on the outcome of my endeavors. Mr. Fouad Ahmad, for all

his helpful and friendly support in the lab work.

To all the colleagues at the Remal Clinic-Central Laboratory, Gaza, where I am

working, for their helping and supporting me.

I would like also to thank all my friends and colleagues, for all of their support and

guidance and encouraging; good luck to all.

Finally, special thanks to my family: father, mother, wife, brothers, sisters, and my

sons. Without their guidance, help and patience, I would have never been able to

accomplish this work.

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Antimicrobial Resistance for Enteric Pathogens Isolated from

Acute Gastroenteritis Patients in Gaza strip, Palestine.

Abstract The antibiotic resistance of enteropathogenic bacteria has profound clinical

implications. Hence, this research was in part an attempt for isolation of

enteropathogenic bacteria and determines their antimicrobial susceptibility patterns.

Samples were collected from acute gastroenteritis patients in Gaza strip, Palestine.

This method of investigation is a matched case-control study.

A total of 132 stool samples were collected from Palestinian patients with acute

diarrhea (gastroenteritis). Among 132 diarrheal patients, 12 (9.1%) of

enteropathogenic bacteria were isolated. Salmonella, Campylobacter coli/jejuni, and

Aeromonas hydrophilia were isolated in equal numbers from samples 3/12 (25%

each), Shigella 2/12 (16.7%), Yersinia enterocolytica 1/12 (8.3%). The two Shigella

spp. are Shigella boydii. The antimicrobial profile of twelve isolated

enteropathogenic bacteria showed high resistance rates for Campylobacter coli/jejuni

(52.4%), followed by Aeromonas hydrophilia (49.2%), Yersinia enterocolytica

(42.9%), Shigella (26.2%) and Salmonella spp. (22.2%). Children younger than 5

years old are more susceptible to infectious diarrhea; in addition, diarrhea is more

frequent in crowded houses with low room's number, and among peoples living in

houses rearing poultry, and pigeons.

Findings from this study demonstrated that Campylobacter coli/jejuni, Aeromonas

hydrophilia, and Yersinia enterocolytica, which are not screened during routine

examinations of stool samples in Palestinian health laboratories in Gaza strip, were

significant enteropathogens in the studied patients. Therefore, we recommend

improving of laboratories in Gaza strip to increase their ability to isolate all types of

enteropathogenic bacteria especially those, which have not isolated routinely in these

laboratories, which require special techniques.

Keywords: Gastroenteritis, Diarrhea, Enteropathogenic bacteria, Antibiotic

resistance, Gaza strip.

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مقاومة البكتیریا المعویة المعزولة من مرضى االلتھابات المعویة الحادة للمضادات الحیویة .فلسطین، ةفي قطاع غز

الملخص

ھذا البحث عزل البكتیریا أھداف من لكلذ، مقاومة البكتیریا المعویة للمضادات الحیویة لھا نتائج طبیة سیئة

.لحیویةالمعویة الممرضة وفحص مقاومتھا للمضادات ا

ھذا البحث تم إجراءه باستخدام . فلسطین، تم جمع العینات من مرضى االلتھابات المعویة الحادة في قطاع غزه

.مرضى وعینات ضابطھ

سھال إ مریض 132 بین من . عینھ من مرضى فلسطینیین یعانون من التھابات معویة وإسھال حاد 132تم جمع

ایروموناس ھیدروفیال ، الكمبیلوباكتر، السلمونیال :ویة وھى كالتاليمن البكتیریا المع%) 9.1(12تم عزل

یرسینیا انتیروكولیتكا ، %)16.7(2/12الشیجال ثم تال ذلك، %)25(3/12عزلت بعدد متساوي

.الشیجال االثنتین ھما شیجال بودیاى %).8.3(1/12

: كالتالي مقاومتھا للمضادات الحیویة یا المعزولة نسبھ عالیھ منالمضادات الحیویة للبكتیر صفح اظھر

یرسینیا انتیروكولیتكا ،%)49.2(یتبعھا ایروموناس ھیدروفیال ، %)52.4(جیجینى /كوالىالكمبیلوباكتر

%). 22.2(السلمونیال ، %)26.2(الشیجال ،%)42.9(

أكثر اإلسھالباالضافھ إلى أن ، باإلسھالصابھ عرضھ لال أكثرعن خمس سنوات أعمارھمالذین تقل األطفال

في حمام أو واجند یربون وبین الناس الذین، تحتوى على عدد غرف اقل التي المزدحمةالبیوت فيانتشارا

.منازلھم

یرسینیا ،دروفیالایروموناس ھی، الكمبیلوباكتر نتائج ھذه الدراسة تظھر انھ تم عزل بعض البكتیریا المعویة مثل

علما أن ھذه األنواع ال تعزل من مزارع البراز في مختبرات قطاع غزه وھى من البكتیریا ، انتیروكولیتكا

لذلك نحن نوصى بتطویر المختبرات في قطاع غزه حتى تستطیع أن تعزل جمیع أنواع .المھمة مرضیا

حیث تحتاج إلى ، رات أن تعزلھا بطریقھ روتینیھالبكتیریا المعویة وخاصة البكتیریا التي ال تستطیع المختب

. تقنیات خاصة

. قطاع غزه، المقاومة للمضادات الحیویة، البكتیریا المعویة، اإلسھال ، االلتھابات المعویة :الكلمات المفتاحیھ

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CHAPTER I

INTRODUCTION

1.1 Overview

Antibiotic resistance was reported very early in the development of these wonder

drugs. Sir Alexander Fleming’s original report in 1929 noted that some bacteria,

including the microbe now called Escherichia coli, were resistant to the effect of

penicillin. In 1940, Edward Abraham and Ernst Chain reported the presence of an

enzyme in E. coli that destroyed penicillin, this was several years before the drug

became widely used to treat patients. In the subsequent decades, bacterial antibiotic

resistance has become a widespread and well-known phenomenon [1].

Inappropriate prescription of antibiotics prompted resistance and increased infectious

disease mortality not only in developing countries but also in developed countries.

Aging populations, changes in behavior and a decline in the development of new

antibiotics exacerbated a deteriorating situation [2].

The antibiotic resistance of enteric bacteria has profound clinical implications

because it threats the life and causes many of serious diseases such as acute

gastroenteritis [3].

Acute gastroenteritis is a severe infection of the gastrointestinal tract (GI).

Sometimes people refer to it as “stomach flu” which is characterized by diarrhea,

stomach pain, nausea, vomiting, fever or feeling unwell. Symptoms may start quite

slowly or come on suddenly. Gastroenteritis usually passes in less than 24 hours but

can continue for several days [4].

Diarrhea is defined as having loose or watery stools at least three times per day, or

more frequently than normal for an individual. Though most episodes of childhood

diarrhea are mild, acute cases can lead to significant fluid loss and dehydration,

which may result in death [5].

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Acute gastroenteritis or infectious diarrhea is one of the leading causes of illnesses

and death in infants and children throughout the world, especially in developing

countries. This is so in Asia, Africa and Latin America, where an estimated 2.5

million deaths occur each year in children less than 5 years of age [6,7]. Diarrhea and

gastroenteritis is also one of the leading causes of deaths among the infants in

Palestine, which constituted about 0.6% in 2003 [8].

Worldwide, the most common pathogens that cause acute gastroenteritis are:

Salmonella spp., Shigella spp., Campylobacter spp., E. coli O157:H7, Listeria

monocytogenes, Vibrio cholerae, Yersinia enterocolitica, Rotavirus,

Cryptosporidium spp., Entamoeba histolytica, and Giardia lamblia. These pathogens

can cause potentially serious diseases, which may be fatal, especially in children. The

common route of infection by these pathogens is the ingestion of contaminated foods

and drinks [9].

Only Salmonella spp. and Shigella spp., are routinely investigated through routine

culture in Gaza strip [10]. Other potential pathogens (such as Campylobacter spp.,

Yersinia spp., Aeromonas spp.), however, are not routinely diagnosed. This often

causes misdiagnosis, and physician overlooks other pathogens, and thus

epidemiological data are inaccurate. Data from the health laboratories all over

Palestine showed that the detection rate of Shigella spp. is very low (about 3 cases in

2009) and even lower for Salmonella spp. (2 cases in 2009) [11].

Moreover, data concerning cases of Campylobacter, Yersinia, Aeromonas and their

relation to infection in Palestinian children are extremely scarce [10-13].

This study performed microbiological investigation of some potential pathogens

associated with diarrhea, to characterize the isolates, their antibiotic resistance and

the epidemiological factors related to the diarrheal disease in patients from Gaza,

Palestine.

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1.2 Objectives:

1.2.1General objective:

To determine antimicrobial resistance for enteric pathogens isolated from acute

gastroenteritis patients in Gaza strip, Palestine.

1.2.2 Specific objective:

1. To detect and identify enteric bacterial pathogens (Salmonella spp., Shigella spp.,

Campylobacter spp., Yersinia enterocolitica, Aeromonas hydrophilia) in fecal

samples from diarrheal patients.

2. To assess the antibiotic susceptibility of isolated bacteria.

3. To investigate possible risk factors for contracting diarrhea.

1.3 Significance:

Poor sanitation and restriction to water access may favour the spread of

communicable diseases, especially infectious diarrhea, which is one of the leading

causes of morbidity in the Gaza strip. In addition, it is one of the more frequent

reasons for primary health care centre (PHCC) attendance in addition to respiratory

tract infections. Infectious diarrhea affects mainly the children who are at risk of

complications, especially when they suffer from malnutrition, which is common in

Palestinian children.

Uncontrolled use of antibiotics due to the lack of antibiotic prescribing policy in

diarrheal infections and for any kind of suspected infection has lead to the emergence

of bacterial resistance, which has become a major concern in Gaza [14-16].

Moreover, there is no available data about the spread and antibiotic resistance of the

enteropathogenic bacteria. Hence, this research was in part an attempt to determine

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the occurrence of antimicrobial resistance for enteric pathogens isolated from acute

gastroenteritis patients in Gaza strip, Palestine.

The results of this investigative work would shed light on this issue and would in part

provide data that may prove useful for health authorities in planning properly to

reduce the incidence of infectious diarrhea and will also aid in determining the

components of the routine stool culture test.

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CHAPTER II

LITERATURE REVIEW

2.1 Acute gastroenteritis

Acute gastroenteritis is a common childhood disease and causes significant economic

burden to developed countries. Children in developing countries are at particular risk

of both morbidity and mortality. Worldwide, gastroenteritis affects 3 to 5 billion

children each year, and accounts for 1.5 to 2.5 million deaths per year or 12% of all

deaths among children less than 5 years of age [17-20]. In developed countries, such

as the United States, acute gastroenteritis seldom causes deaths, however, it still

accounts for 300 deaths per year. Moreover, it puts a heavy burden on the health care

system. Acute gastroenteritis causes 1.5 million visits to primary care providers each

year and 220,000 hospital admissions for children under the age of 5 years; that is

10% of all the hospital admissions of children in the United States [18]. In general,

developing countries have a higher rate of hospital admissions as compared to

developed countries. This may be due to the facts that children in developed

countries have a better nutritional status and better primary care. The difference can

also be explained by the fact that, the incidence of acute gastroenteritis is

significantly higher in developing countries than in industrialized countries [21].

2.2 Antimicrobial resistance

Antimicrobial resistance is one of the world’s most serious public health problems,

many of the microbes (bacteria, viruses, protozoa) that cause infectious disease no

longer respond to common antimicrobial drugs. The problem is so serious that unless

concerted action is taken worldwide, we run the risk of returning to the pre-antibiotic

era when many more children than now died of infectious diseases. The major

infectious diseases kill over 11 million people per year. The prevalence rate of

antimicrobial resistance all overall the world of diarrheal shigellosis is 10-90% for

ampicillin and 5-95% for trimethoprim/sulfamethoxazole [22].

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For this reason the microbial antibiotics resistance is receiving increasing attention in

light of the increasing incidence of human bacterial infections resistant to antibiotic

treatment [23]. The resistance of enteropathogenic bacteria to commonly prescribed

antibiotics is increasing both in developing as well as in developed countries;

resistance has emerged even to newer, more potent antimicrobial agents [24].

2.3 Diarrheal disease is the most common cause of illness

In 2004 diarrheal disease affects far more individuals than any other illness, even in

regions that include high-income countries. About 4620.4 million people affected

with diarrheal disease. In addition, diarrhea is the second most common cause of

child deaths worldwide as showed in figure (1.2) [25].

causes of death among children aged under five years, 2004

Pneumonia17%

Diarrhea16%

Other13%

Malaria7%Measles

4%

Aids2%

Injuries4%

Neonatal causes

37%

Figure (1.2): Distribution of causes of death among children aged under five years,

2004 [25].

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2.4 Common bacterial causes of diarrhea

Many types of enteropathogenic bacteria cause acute gastroenteritis and these types

are different according to regions. In the following section, we will discuss the

isolated enteropathogenic bacteria in this study.

2.4.1 Salmonella spp. history

Salmonella first identified in 1885. It was named for one of the two men who

discovered it, Daniel Elmer Salmon (the other was Theobald Smith). They first found

Salmonella in hogs that were ill with a disease called hog cholerae [26].

Salmonella is a gram-negative rod, facultative anaerobic, flagellated bacterium, has a

growth rate, or division rate, of 40 minutes, prefers to grow at 37°C, but has the

ability to grow at a wide range of temperatures, from 6 to 46°C. This provides

Salmonella with many opportunities to grow. Optimum growth occurs at a pH of 6.5-

7.5. It is the pathogenic agent of salmonellosis, a major cause of enteric illness and

typhoid fever, leading to many hospitalizations and a few rare deaths if no antibiotics

are administered. Salmonella outbreaks are linked to unhygienic food preparation,

cooking, reheating and storage practices. The bacterium can be isolated from raw

meat and poultry products as well as from milk and milk-based products [26,27].

Salmonella is a ubiquitous human and animal pathogen. This genus contains > 2,500

recognized serotypes and is divided into two species, Salmonella bongori and

Salmonella enterica. Salmonella enterica consists of six subspecies (i.e., enterica,

salamae, arizonae, diarizonae, houtenae, and indica) [28]. Serovars of the bacterium

Salmonella enterica subsp. enterica are known to be a significant cause of foodborne

illness in humans. It is well established that different serovars can be associated with

different animals e.g., S. enteritidis in poultry, which act as a source of foodborne

infection in humans [29].

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Salmonella enterica subsp. enterica serotypes can also be divided into subdivisions

according to their host adaptation. For example Salmonella serotypes can be divided

into (i) host-restricted Salmonella serotypes (i.e., serotypes exclusively associated

with one particular host, e.g., Salmonella Typhi and Paratyphi A); (ii) host-adapted

Salmonella serotypes (i.e., serotypes prevalent in one particular host species, but able

to cause disease in other host species, e.g., Salmonella Choleraesuis); and (iii)

unrestricted Salmonella serotypes (i.e., serotypes capable of causing self-limiting

gastroenteritis and, less commonly, systemic disease in a wide range of host species,

e.g., Salmonella Typhimurium). Salmonella Paratyphi A also causes typhoid fever,

although the symptoms are typically milder than those caused by Salmonella Typhi

[30].

Non-typhoidal Salmonella serotypes are responsible for gastroenteritis in humans

and other animals. These serotypes are mainly transmitted by ingestion of food, or

water contaminated with infected feces [31], but can also be transmitted by direct

contact [32,33]. Disease caused by non-typhoidal Salmonella is one of the most

common bacterial foodborne diseases worldwide [34]. Salmonella Typhimurium is

one of the most common non-typhoidal Salmonella serotypes, is found worldwide,

and can cause disease, predominantly self-limiting gastroenteritis, in a large number

of animal species [30]. Because of the abuse of antimicrobial drugs, the antibiotic

resistance of Salmonella strains from humans is expected to increase. It has been

reported that there has been an increase in antibiotic resistance of Salmonella isolates

from humans [35].

The detection of Salmonella therefore remains a highly important issue in

microbiological analysis for food safety and standards [36], and to guide clinicians in

the diagnosis of enteric pathogens [37].

2.4.2 Shigella spp. history

Kiyoshi Shiga discovered shigella during an outbreak in Japan in 1897, which

investigated 36 shigellosis patients and identified the bacilli as the etiology agent in

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1898 [38]. To acknowledge him for his discovery, the 1930 Edition of Bergey’s

Manual of Determinative Bacteriology formally renamed the genus as ‘Shigella’

[39].

Shigella is gram-negative rods, non-motile, belonging to the family

Enterobacteriacae. The genus Shigella includes four species: S. dysenteriae, S.

flexneri, S. boydii and S. sonnei designated groups A, B, C and D, respectively. All

species of Shigella cause acute bloody diarrhea by invading and causing patchy

destruction of the colonic epithelium. This leads to the formation of micro-ulcers and

inflammatory exudates, and causes inflammatory cells (polymorphonuclear

leucocytes, PMNs) and blood to appear in the stool. The diarrheal stool contains 106-

108 Shigellas per gram. Environmental conditions like dryness or direct exposure to

sunlight may lead to the death of the organism due to its sensitivity. The first three

species include multiple serotypes. S. sonnei and S. boydii usually cause relatively

mild illness in which diarrhea may be watery or bloody. S. flexneri is the chief cause

of endemic shigellosis in developing countries [40], while S. sonnei is the most

commonly isolated species in developed countries, representing over 70% of the total

isolates in the United States of America [41] and 87% in occupied Palestine [42].

Shigellosis is an ongoing global public health problem. Due to the fecal-oral

transmission route of the organisms, the overwhelming burden of shigellosis is found

in resource-poor settings with inadequate sanitation [43,44]. With an estimated

number of episodes exceeding 90 million per annum in Asia alone, shigellosis

represents a significant proportion of the total number of bacterial gastrointestinal

infections worldwide [45]. Unlike other related bacteria which can cause a particular

disease syndrome in specific locations (e.g. Salmonella Typhi) [46], it is a disease

which "bridges the gap" between industrialized and developing countries. A report

from the National Centre for Infectious Diseases in the United States of America

found the incidence of shigellosis to be 7.6 cases per 100,000 persons in 1993 [41].

Shigella species the causative agent of bacillary dysentery (shigellosis) are highly

adapted human pathogens that are capable of invading and colonizing the intestinal

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epithelium, which results in severe inflammatory colitis [47]. Shigellosis symptoms

includes fever, headache, malaise, anorexia and occasionally vomiting, followed by

excretion of profuse watery diarrhea proceeding bloody and/or mucoid diarrhea [48].

All the members of the genus Shigella are pathogens restricted to infecting humans

and exert their effects on the gastrointestinal mucosa via the production of a

multitude of virulence factors, including enterotoxins and effector proteins [49,50].

Prompt treatment of shigellosis with appropriate antimicrobial agents not only

shortens the duration and severity of the illness but also reduces microbial carriage

and thus spread of infection in the community, but unfortunately emergence of

antimicrobial resistance has complicated the empirical therapy for treatment of

shigellosis, due to the prevalence of antimicrobial resistance to some antibiotics used

to treat Shigella and this resistance differs within Shigella serogroups [51,52].

The antibiotics recommended by the World Health Organization (WHO) include

ciprofloxacin, ceftriaxone and pivmecillinam, which are effective in reducing the

clinical and bacteriological signs and symptoms of dysentery, and thus can be

expected to decrease diarrhea mortality attributable to dysentery [53].

Shigella can be isolated from stool samples by culturing it on specific media, and

simple conventional biochemical's tests are used to identify Shigella species, which

grouped serologically by slide agglutination with specific antisera. Antibiotic

susceptibility is usually determined in all samples by disc diffusion method

according to the Clinical and Laboratory Standards Institute (CLSI) [54,55].

2.4.3 Campylobacters spp. history

The first description of Campylobacter in the stools of diarrheic children was

observed by Escherichia in 1886 in Germany and their first isolation was performed

by McFadyean and Stockman in 1913 [56]. Up until the middle of the 1940’s, these

bacteria were named Vibrio fetus for those isolated from some animals (ovines,

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bovines and porcines) abortions, or Vibrio jejuni and Vibrio coli for those isolated

from feces of these animals [57]. The probable implication of these microbes in

infectious disease of food origin was established in 1946 when Levy described a

gastroenteritis epidemic in a prison population in Illinois in the United States [58].

Until 1963, these microaerophilic vibrios were included in the Vibrio genus. At this

time, Sebald and Veron proposed the creation of the Campylobacter genus

(etymologically from the Greek kampulos = curved, bacter = rod) [59].

Campylobacter is gram-negative, spiral-shaped, has a single polar flagellum, motile,

non spore forming, sometimes capsulated, microaerophilic bacterium i.e. grow in the

presence of an atmosphere poor in oxygen, generally between 5 and 10 %. Many

authors find that Campylobacter is more capnophilic than microaerophilic, i.e. that it

requires an atmosphere enriched in CO2 to proliferate (generally 10%). Most often,

but not exclusively, the gaseous mixture appropriate for culture of the microbe is: O2

= 5%; CO2 = 10%; N2 = 85%. All species of the Campylobacter genus can

proliferate at 37°C and are true mesophiles, but grow beast from 37°C to 41.5°C

(Campylobacter coli, jejuni, lari grow beast at 41.5 but usually not Campylobacter

fetus) and its optimal pH zone is from 6.5 to 7.5, presence of 0.5 % sodium chloride

(NaCl) in the medium is recommended for culture, whereas concentrations exceeding

1.5 % tend to inhibit growth [57,60].

Worldwide, Campylobacter is recognized as the major cause of gastroenteritis and

diarrheal disease in humans [61-64]. The majority (approximately 90%) of cases of

Campylobacter gastroenteritis in humans is caused by Campylobacter jejuni, and

most of the remainder is caused by Campylobacter coli [65]. Poultry, particularly

chickens, account for the majority of human infections caused by Campylobacter

[66,67]. Campylobacter jejuni and Campylobacter coli are the most prevalent species

[62,65,68].

Campylobacteriosis, an acute bacterial enteritis which is a major problem worldwide,

mostly caused by Campylobacter coli and Campylobacter jejuni. These pathogens

live in the intestinal tract of most avian species [69].

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Outbreaks of campylobacteriosis are relatively rare; most infections seem to be

sporadic [70]. However, the epidemiology of sporadic campylobacteriosis, especially

the routes of transmission, is to a great extent unclear [71,72]. Internationally it is

estimated that handling, preparation and consumption of broiler meat may account

for 20% to 30% of the human cases, however, it has been suggested that chicken as a

reservoir might account for between 50% and 80% of the cases [73].

Campylobacteriosis cases might also be caused by other risk factors than

consumption and handling of poultry meat like as broiler farms could contaminate

the environment with further spread to new broiler farms or to humans living in the

area and local environmental factors, such as climate [74].

Campylobacter gastroenteritis is a self-limited disease, and antimicrobial therapy is

not generally required; however, treatment can decrease the duration and severity of

illness if it is initiated early in the course of infection [75]. Infants, elderly people,

and immunocompromized individuals are at higher risk of developing more severe

campylobacteriosis. In these cases, early antibiotic therapy is especially effective

[76].

The primary drugs of choice for treatment of human campylobacteriosis include

erythromycin and ciprofloxacin. Resistance to ciprofloxacin and erythromycin in

Campylobacter recovered and occurred more frequently among C. coli than C. jejuni

[77].

Rapid and appropriate identification of the etiologic agent of infectious

gastroenteritis is important, since there are major differences in the treatments

required for the different agents. C. jejuni and C. coli in human stool samples are

commonly detected by a bacterial culture test. The test procedure includes culturing

of the stool on selective agar medium and the subsequent identification of

Campylobacter-suspected colonies. Several another methods, including a

commercially available enzyme immunoassay [78], polymerase chain reaction

(PCR)-based assays [79], and a DNA hybridization-based assay [80], have already

been reported. Although these assays have excellent sensitivity, they require a

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comparatively complicated and time-consuming procedure. Their simplicity and

rapidity appear to be insufficient for routine or emergency use in the microbiology

laboratory [81].

2.4.4 Yersinia enterocolitica history

Since the causative agent of plague was first identified as a gram-negative bacterium

by Alexander Yersin in 1894, our understanding of the epidemiology, genetics and

evolution of Yersinia has come a long way [82]. Yersinia was classified in the

Pasteurella genus until 1944 when Vanloghon reclassified them as Yersinia genus in

family Enterobacteriaceae. Despite similarities with other Enterobacteriaceae

members, slight differences are exist such as slow growth on solid media, colonies of

at most 1mm in diameter, and better growth at cooler temperature [83,84]. Metchock

et al., reported yersiniosis most commonly in cooler months [85].

Yersinia spp. is gram-negative rods or coccobacilli with bipolar staining that belong

to the Enterobacteriaceae family [86]. Eleven species are known, but only 3 are

important human pathogens; Y. enterocolitica is the most common of these, while Y.

pseudotuberculosis is less frequent, and Y. pesties is rare [87].

Y. enterocolitica is a zoonotic bacterium causes a variety of gastrointestinal disease

in humans (Yersiniosis). Clinical symptoms of yersiniosis first appear after an

incubation period of about 5 days and include diarrhea, fever, vomiting, tenesma and

abdominal pain. In older children and young adults, abdominal pain in the right

lower abdomen can occur, which may be mistaken for appendicitis

(pseudoappendicitis). Typically, symptoms disappear within 1-2 weeks after onset.

Sequelae such as reactive arthritis or erythema nodosum sometimes occur. Y.

enterocolitica infections are usually sporadic, although outbreaks have been reported.

Y. enterocolitica species can be isolated from a variety of domestic and wildlife

animals, e.g., pigs, cattle, sheep, goats, dogs, cats, wild boars, and small rodents [88-

90]. Thought to be primarily transmitted to humans via ingestion of contaminated

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foods, water and milk or ingestion of uncooked meat products, especially pork

[88,91]. However, other risk factors, such as pet animal contact, have been reported

[92]. The majority of cases of enterocolitis are seen in children aged 1-4 years

[93,94]. Moreover, these infections show a modest predilection for males, with male

to female ratio of 1.7:1 [95].

Currently, Y. enterocolitica is represented by six biovars (1A, 1B, 2, 3, 4 and 5) and

more than 30 distinct serovars. The virulence of known pathogenic biovars namely

1B and 2-5 is attributed to PYV (plasmid for Yersinia virulence) plasmid and

chromosomally borne virulence factors [96].

The species encompasses both pathogenic strains including bio/serotypes 4/ O:3 and

2/O:9 and strains which are classically considered non-pathogenic, namely strains of

biotype (BT) 1A or non-biotypable strains. However during study in Finland carried

from January 1st 2006 to December 31st 2006 in 10 clinical microbiology laboratories

showed that the majority of YE strains isolated from Finnish patients belonged to BT

1A. The symptoms and sources of patients with Y. enterocolitica biotype (BT) 1A

(YE BT 1A) were different from the pathogenic YE. In addition, BT 1A patients had

more unspecific complaints and protracted gastrointestinal disorders, which suggest

that the original cause of the illness may have been other than YE BT 1A. This is

supported by the fact, that they did not find any YE BT 1A strains in small children.

However, it is still possible that in the highly heterogeneous group of YE BT 1A

there are subgroups that can cause a disease, but this needs further investigations.

While the significance of the YE BT 1A strains remains unclear, the correct

registering of infections caused by the truly pathogenic YE strains is essential for

health protection purposes [97].

Yersiniosis contributes substantially to foodborne diseases in industrialized countries

and is therefore notifiable through national surveillance systems in most countries

within the European Union (EU) [98]. The incidence of yersiniosis is reported to be

10%-30% in European countries but its a rare disease in Muslim countries (0.06%-

2%) due to the scarcity of pork consumption [83,84].

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Y. enterocolitica isolates were completely resistant to ampicillin, penicillin,

cephalotin, and erythromycin [99]. One of the purposes of this work is to investigate

the presence, isolation, and antimicrobial susceptibility of Y. enterocolitica from

stool samples of diarrheal patients by microbiological methods using cold-enriched

media in order to improve the chances of recovering pathogenic Y. enterocolitica.

2.4.5 Aeromonas hydrophila history

Aeromonas spp. was first isolated from water and diseased animals over 100 years

ago. Originally, A. hydrophila was identified as one of four Aeromonas species by

Popo (1984) and he placed it in the family Vibrionaceae [100]. The genus

Aeromonas currently belonging to the family Aeromonadaceae that includes a group

of 21-gram negative bacterial species (one of them is A. hydrophila). A. hydrophila is

a pathogenic bacterium, widely distributed in the environment. Although it is clear

that members of this genus are primarily aquatic organisms, they can colonize other

habitats and cause infections in invertebrates and vertebrates [101,102]. Aeromonas

(principally A. hydrophila) currently has the status of a foodborne pathogen of

emerging importance [103,104].

Members of the genus Aeromonas are gram negative rods, facultative anaerobic,

non-spore-forming, motile by a single polar flagellum, catalase-positive, oxidase-

positive, its neither salt (< 5%) nor acid (min. pH ~ 6.0) tolerant and grows optimally

at around 28°C. It has the ability to grow at cold temperatures, reportedly as low as

0.1°C for some strains. It can be metabolize glucose by both the respiratory and

fermentative pathways and show resistance to the vibriostatic agent O/129. It can be

divided into two groups: the first includes the psychotrophic Aeromonas, represented

by Aeromonas salmonicida and the second, by the mesophillic Aeromonas. It

principal reservoir is the aquatic environment such as fresh water lakes and streams

and waste water systems [101, 102, 105].

Von Graevenitz and Mensch (1968) reported associations of aeromonads with human

disease in a review of 30 cases of Aeromonas infection or colonization, providing

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evidence for their recognition as human pathogens and suggesting that some

aeromonads may be associated with gastrointestinal disease [106].

In addition, Kirov (2003) notes the potential role of A. hydrophila in human

gastrointestinal infection. It is able to form lateral flagella on solid surfaces and able

to produce factors associated with gastroenteritis. These properties are exoproteases,

exotoxins, cytotoxins, endotoxins, siderophores, invasins, adhesions, S-layers and

flagella [107,108].

Gastrointestinal infections of Aeromonas spp. are generally considered waterborne,

for this reason, A. hydrophila has been placed on the United State of America (USA)

protection Agency Contaminant Candidate List of emerging pathogens in drinking

water [109].

A. hydrophila causes diarrheal infections, most commonly in children's (especially

those under 5 years) and in immunocompromised patients. The organism is being

isolated lately with high frequency from patients with traveller's diarrhea [110, 111].

There has been an increasing incidence of antimicrobial resistance among

Aeromonas spp. from both environmental and clinical sources [112,113]. A study in

2006 observed antibiotic resistance in 48% of the Aeromonas isolated from oysters

[114].

So the Food and Agriculture Organization / World Health Organization

(FAO/WHO) commission recommends that to prevent waterborne disease in

developing countries, aquatic environments directly impacting human populations

should be characterized physically, chemically and microbiologically [115].

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2.5 Epidemiology and antimicrobial resistance of gastroenteritis

pathogens

In a local study in El-naser hospital from 1999 to 2006, the results showed that the

isolation frequency was 65/3570(1.8%) for Salmonella spp. and 28/3570(0.8%) for

Shigella spp., Shigella flexneri (16/28) was the most frequently isolated Shigella spp.

Most of the Shigella isolates were resistant to trimethoprim-sulfamethoxazole (89%),

ampicillin (79%) and chloramphenicol (46%), whereas most of the Salmonella

isolates showed resistance to ampicillin (62%), trimethoprim-sulfamethoxazole

(35%), chloramphenicol (35%) and cephalexin (26%) [116].

In an Egyptian study on 714 patients with diarrhea, they found that 14% were

Enterotoxigenic Escherichia coli (ETEC)-associated diarrhea, 1.0% Campylobacter-

associated diarrhea and Shigella-associated diarrhea represented 2%. Children with

Shigella or Campylobacter-associated diarrhea were reported as watery diarrhea and

rarely dysentery. ETEC did not have any clinically distinct characteristics [117].

In a study in Jordan investigated the polymicrobial infections in 220 children with

diarrhea in a rural population, potential pathogenic agents isolated from 143(65%)

children were identified by molecular and standard microbiological methods. Co-

infections with two or more agents were detected in 50(35%) of cases. Escherichia

coli, Shigella spp., Giardia and Entamoeba histolytica were found to be predominant

[118].

Another study in Jordan investigated the enteropathogens associated with cases of

gastroenteritis in a rural population in 180 children. Pathogens and potential

enteropathogens were identified in 140(77.8%) of the patients, with more than one

pathogen being recovered from 67(37.2%) of the patients. Potentially pathogenic

parasites were observed in 90(50%) patients; those that were associated significantly

with diarrhea were Giardia lamblia, Blastocystis hominis, Cryptosporidium spp.,

Entamoeba histolytica and Cyclospora cayetanensis. Pathogenic bacteria were

isolated from 72(40%) patients, and, of these, 62.5% were resistant to at least one

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antibiotic, and 30.6% of these were multidrug resistant. Diarrheagenic Escherichia

coli strains were found in 14.3% of the samples. The most common enteropathogenic

bacteria found were Shigella spp., Campylobacter jejuni and Y. enterocolitica [119].

Analysis of 1967 Non typhoidal salmonella (NTS) isolated from clinical samples

between 1990 and 2006 at the AgaKhan University (AKU), a tertiary care hospital in

Karachi, Pakistan, showed a significant increase in ciprofloxacin resistance from

23% in 2002 to 50.5% in 2006, with increased mean minimum inhibitory

concentration (MIC) values from 0.6 to 1.3 µg/mL. Ceftriaxone resistant NTS also

increased and Extended spectrum beta-lactamase (ESBL) production was seen in

98.7% isolates. These isolates exhibited high resistance against

amoxicillin/clavulanic acid (57%), gentamicin (69%), amikacin (44%) and

piperacillin/tazobactam (30%). No resistance to carbapenem was seen. Ceftriaxone

resistance was significantly higher in children < 1 year, in invasive isolates and in

Salmonella Typhimurium [120].

A study was conducted to determine the etiology of diarrhea in a hospital setting in

Kolkata, India. Active surveillance was conducted for 2 years on two random days

per week by enrolling every fifth diarrhea patient admitted to the Infectious Diseases

and Beliaghata General Hospital in Kolkata. Most of the patients (76.1%) had acute

watery diarrhea in association with vomiting (77.7%) and some dehydration (92%).

Vibrio cholerae O1, Rotavirus and Giardia lamblia were the important causes of

diarrhea. Among Shigella spp., S. flexneri 2a and 3a serotypes were most

predominantly isolated. Enteric viruses, Enteropathogenic Escherichia coli (EPEC)

and Enteroaggregative Escherichia coli (EAEC) were common in children <5 year

age group. Atypical EPEC was comparatively higher than the typical EPEC,

multidrug resistance was common among V. cholerae O1 and Shigella spp. including

tetracycline and ciprofloxacin. Polymicrobial infections were common in all age

groups and 27.9% of the diarrhea patients had no potential pathogen [121].

Other study was carried out to detect the bacterial agents associated with bloody

diarrhea in children and to determine their antimicrobial susceptibility patterns

between June 2001 and January 2008. 249 children with bloody diarrhea were

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studied, Shigella and Shiga toxin-producing Escherichia coli (STEC) were recovered

from 48(19.3%) and 3(1.2%) of the total of cases, respectively. In 49 out of 249

children, in whom other enteropathogens were investigated, recovered

Campylobacter jejuni from 7 children (14.3%), Salmonella spp. from 2(4.1%) and

Aeromonas spp. from 1(2%) in addition to Shigella from 7 children (14.3%). Thirty-

four (70%) Shigella isolates showed resistance to ampicillin and 13(27%) to

trimethoprim-sulfamethoxazole. All Shigella isolates were susceptible to nalidixic

acid, ciprofloxacin and ceftriaxone. Salmonella and STEC isolates were susceptible

to all antibiotics assayed. Thus, the use of trimethoprim-sulfamethoxazole or

ampicillin would not be appropriate for the empirical treatment of Shigella -

associated diarrhea [122].

A study was carried in Sofia, for the period from 1987 to 2008 to detect

Campylobacter jejuni and coli from diarrheal patients. The study included patients

from 0 to over 65 years old. A total of 51,607 faecal samples were screened for

Campylobacter. C. jejuni and C. coli were detected in 1847(3.58%) of the strains,

with the highest percentage in 1988 (7.5%) and the lowest in 2006 (0.3%).

Campylobacteriosis occurred most frequently in the wet months of March, April,

May and June, with 105, 102, 124 and 141 cases, respectively, and was rare in

January with 25 cases. The most affected groups were children between 0 and 4

years of age (52%) and between five and 14 years of age (30%). Campylobacter

infection occurred in 22% of all bacterial gastrointestinal diseases in the city of Sofia

during the study period. Salmonella was the most frequently identified pathogen with

32%, followed by Shigella (30%), Campylobacter (22%) and diarrheagenic

Escherichia coli (16%). The study shows that Campylobacter plays an important role

as a bacterial cause of enterocolitis in Sofia, Bulgaria [123].

Two hundred fifty children presenting with diarrhea at 2 teaching hospitals in Mosul,

Iraq over a 9-month period were studied for the presence of Yersinia spp. in stools by

cold-enrichment culture at 4°C for 21 days. Yersinia spp. was isolated from the

stools of only 4 patients; 3 isolates were identified as Y. enterocolitica and 1 was Y.

pseudotuberculosis. One Y. enterocolitica isolated from one case by blood culture.

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The antibiogram test for the isolates was determined. Cross-reaction between Y.

pseudotuberculosis and Salmonella Typhi or S. Paratyphi B and between Y.

enterocolitica and Brucella was detected serologically [97].

From August 2005 to August 2007, researchers were conducted a prospective cohort

study among patients ≥ 18 y hospitalized with community-acquired gastroenteritis in

a university hospital in Berlin, Germany. Stool samples were examined for 26

gastrointestinal pathogens, supplemented by serologic tests for antibodies to

Campylobacter spp., Yersinia spp., and Entamoeba histolytica. 104 were included in

the study. A non-infectious etiology was diagnosed in 8 patients (8%). In 79 (82%)

of the remaining 96 patients at least one microorganism was identified,

Campylobacter spp. (35%) was detected most frequently, followed by Norovirus

(23%), Salmonella spp. (20%), and Rotavirus (15%). In 46% of the patients with

Campylobacter spp. infection, the diagnosis was made solely by serology. More than

one pathogen was found in seventeen (22%) patients [37].

In one study, which was carried out, to determine the prevalence of five

enteropathogen diarrheogenic agents in Mexico City from September 2004 through

December 2006, all diarrheal samples were positive for one or more enteropathogens.

The most common enteropathogens in diarrheal samples were E. histolytica / dispar

(70.3%), Salmonella (ohio 28.3%; Typhimurium 16.3%; infantis 8%; anatum 0.6%;

Newport 0.3%), G. intestinalis (33%), E. coli (ETEC 13.3%; EPEC 9.3%;

Verocytotoxin-prtducing E. coli (VTEC) 8.6%; Enteroinvasive E. coli (EIEC) 1%)

and Shigella spp. (flexneri 1.6%, sonnei 1%). Infections by two (24%), three (16%)

and four (12%) pathogens were observed [124].

In the few cases of acute childhood diarrhea that require antimicrobial therapy, the

correct choice of the drug depends on detailed previous knowledge of local strains.

In order to establish such parameters in Salvador, researcher reviewed the results of

all 260 positive stool cultures of children between 0 and 15 years of age during two

years at a pediatric tertiary care facility in Salvador, Brazil. Bacterial strains had been

presumptively identified by culturing in selective media and by biochemical, testing

and their antimicrobial susceptibility patterns were automatically detected. Data

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about patients' sex and age, monthly distribution of the cases, pathogens isolated and

their antimicrobial resistance patterns were recorded. Males corresponded to 55.4%

of their samples, and most of the patients (42.7%) were between one and four years

of age. Shigella was the commonest pathogen, being found in 141(54.3%) cultures,

while Salmonella was found in 100 (38.4%) cultures and EPEC in 19(7.3%).

Salmonella was the main causal agent of diarrhea in children younger than five years

old, whereas Shigella was the most frequent pathogen isolated from the stools of

children between five and 15 years old. The peaks of incidence correspond to the

periods of school vacations. Shigella presented a very high resistance rate to

trimethoprim-sulfamethoxazole (90.1%) and to ampicillin (22.0%), while Salmonella

presented very low resistance rates to all drugs tested [125].

One study was undertaken to report the current antibiotic resistance in common

bacterial enteropathogens isolated in a tertiary care hospital in north India. Stool

samples from 119 (88 male, 31 female) patients yielded Shigella, Salmonella, Vibrio

cholerae and Aeromonas. Fifty two per cent (62/119) of patients were children and

70% were below the age of 5 years old. Twenty-seven patients developed hospital-

acquired diarrhea. Among all enteropathogens, Shigella spp. was the commonest

followed by non-typhoidal Salmonella (27), V. cholerae O1 El tor serotype Ogawa

(19), Aeromonas spp. (14), Salmonella Typhi and S. Paratyphi A (2 isolates each).

Resistance to antimicrobial agents was common among all pathogens. Among

Shigella an overall resistance of 63.6%, 58.1% and 16.3% was observed for nalidixic

acid, trimethoprim-sulfamethoxazole and furazolidone respectively. Seven isolates of

Shigella were resistant to ciprofloxacin, while (18.5%) of non-typhoidal salmonella

were resistant to ciprofloxacin. V. cholerae were generally susceptible to tetracycline

(only 1 isolate out of 13 resistant) and other drugs except nalidixic acid (89.5%

resistance) and trimethoprim-sulfamethoxazole (77.8% resistance) [24].

A study was carried out to document the presence of pathogenic A. hydrophila in

diarrheal stool samples in children at Coimbatore, India. Of the 216 samples, 21

(9.7%) were positive for A. hydrophila. Among them 20 isolates were resistant to

bacitracin. Most of the isolates showed multiple antibiotic resistances. Among the 21

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isolates, protease and haemolysin producers were 100% and 95 % respectively.

About 76% of the isolates produced slime [126].

In Brazil, the prevalence of enteropathogens associated with diarrheal disease in 94

infants < 5 years old was investigated. Cryptosporidium (85.1%) topped the list of

parasite isolates, followed by Entamoeba histolytica (56.4%) and Giardia intestinalis

(4.3%). Four samples contained EPEC (4.3%). Salmonella and Shigella, however,

were not detected, and only one sample contained Rotavirus (1.1%) [127].

In Sao Paulo, Brazil, the etiologic profile of acute diarrhea in 154 children aging less

than 5 years was studied. Intestinal pathogens were detected in 112(72.8%) cases.

The association of two or more intestinal pathogens occurred in 47(30.5%) cases.

The pathogens identified were, Rotavirus; 32(20.8%), bacteria; 53(34.4%), both; 25

(16.2%), and 2(1.4%) with Giardia intestinalis (in one case associated with

Rotavirus and in another one associated with bacteria). Altogether, there were 105

bacterial isolates; 90 were Escherichia coli (EPEC 27, Diffuse adhering E. coli

(DAEC) 24, ETEC 21 and EAEC 18), 12 were Shigella spp., 2 were Salmonella spp.,

and one was Yersinia spp. Children with mixed infections (viral and bacterial) had

increased incidence of severe vomiting, dehydration and hospitalization [128].

The trend in isolation of Vibrio cholerae, Shigella, and Salmonella in neonates with

diarrhea in Bangladesh was investigated. The study population included 240

neonates who were admitted with acute diarrhea and other medical complications to

the inpatient department of The International Centre for Diarrheal Disease Research

(ICDDR) hospital, Dhaka, Bangladesh, in 2001. A single enteric pathogen was

detected in 71(29.5%), and multiple pathogens were detected in 12(5%) of the

neonates. Enteropathogens identified were as follows: V. cholerae O1 (17.5%),

Shigella spp. (9.1%), Salmonella spp. (3.3%), Aeromonas spp. (3.7%), and Hafnia

alvei in (0.8%) of the neonates [129].

In a 12-month period, 561 stool cultures from Yemeni children aged 1-60 months

and presenting with diarrhea, were analyzed to identify the bacterial etiology and

their antimicrobial resistance to the commonly used antibiotics. A total of 190

(33.9%) were positive for bacterial culture. Most of the positive cultures (58%) were

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from children aged 1-12 months. The majority of the positive cultures were EPEC

(58.4%), Salmonella spp., and Shigella spp. (20% each). Campylobacter were found

to be an extremely uncommon agent of childhood diarrhea making only 1.6% of the

positive cultures. The majority of the Salmonella were group C (60.5%) and group B

(29%). Of the Shigella isolates, 13(34%) were S. flexneri, and seven (18%) S.

dysentrea. More than two-thirds of the Salmonella isolates were resistant to nalidixic

acid, chloramphenicol, trimethoprim-sulfamethoxazole, gentamicin, and amoxicillin,

while 42% were resistant to cefotaxime. Most of the Shigella isolates were

susceptible to nalidixic acid and cefotaxime, and resistant to the other antibiotics. All

the tested EPEC isolates were resistant to amoxicillin, 83% were resistant to

trimethoprim-sulfamethoxazole, 62% to chloramphenicol, and 54% to gentamicin,

while only 16 and 6 % were resistant to nalidixic acid and cefotaxime, respectively

[130].

Researchers studied microorganisms associated with infant diarrhea in a group of

256 children admitted to a public pediatric hospital in Montevideo, Uruguay.

Diagnostic procedures were updated to optimize detection of potential pathogens,

which were found in 63.8% of cases, and to be able to define their characteristics

down to molecular or antigenic type. Mixed infection with two or more agents was

detected in more than one-third of positive cases. Escherichia coli, especially

(EPEC), were shown to be prevalent. Rotavirus, Cryptosporidium, Campylobacter

(mainly Campylobacter jejuni), and Shigella flexneri were also often identified.

ETEC, Salmonella, and Giardia lamblia were sporadically recognized. Unusual

findings included two EIEC strains, one Shigella dysenteriae, and a non-O:1 Vibrio

cholerae culture. EPEC bacteria and S. flexneri (but not Salmonella) showed

unusually frequent antimicrobial resistance, especially towards beta-lactam

antibiotics, which is the subject of ongoing work [131].

A group of researcher in Gaborone, Botswana investigated the Shigella and

Salmonella strains isolated from 221 children under 5 years, and their antibiotic

susceptibility patterns. They isolated Shigella and Salmonella from (21%), (3%)

respectively. S. boydii (13%) was the most prevalent Shigella species followed by S.

flexneri (6%) and S. sonnei (2%). Salmonella species were S. Typhimurium and S.

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Paratyphi. Antibiograms of the predominant isolates showed that most Shigella

species were resistant to ampicillin but susceptible to chloramphenicol. The

Salmonella species were susceptible to chloramphenicol, collistin-sulphate,

gentamicin, trimethoprim-sulfamethoxazole, and ampicillin [132].

In a study in Jeddah, Saudi Arabia, investigating the prevalence of viral, bacterial

and parasitic enteropathogens among young children with acute diarrhea, in 576

fecal samples collected from children < 5 years old suffering from acute diarrhea

and attending hospitals and outpatient clinics. One or more enteropathogens were

identified in 45.6% of the stool samples. Mixed infections were detected in 12.2% of

the diarrheal cases. Rotavirus was detected in 34.6% of the samples of the

hospitalized patients and in 5.9% of the samples of the outpatients. Other etiologic

agents recognized included Escherichia coli (13%), of which 3.8% were (EPEC) and

1.9% enterohaemorrhagic E. coli. Other detected pathogens were: Klebsiella

pneumonia (4%), Giardia intestinalis (3.1%), Salmonella spp. (3%), Shigella flexneri

(2.6%), Entamoeba histolytica (2.2%), Trichuris trichiura, Hymenolepis nana, and

Ascaris lumbricoides (0.7% each), and Candida albicans (0.5%) [133].

The prevalence of enteropathogens associated with diarrheal disease in 130 infants

living in the poor urban areas of Porto Velho, Brazil, was investigated. 80% of

diarrheal cases were observed in the groups under 2 years of age. Rotavirus (19.2%)

was the most frequent enteropathogen associated with diarrhea, followed by Shigella

flexneri (6.15%) and S. sonnei (1.5%) and Salmonella spp. (6.9%). ETEC infections

(3.1%), EPEC (2.3%), EIEC (0.8%) and Y. enterocolitica (0.8%). Mixed infections

were frequent, associating Rotavirus, EPEC and Salmonella spp. with Entamoeba

histolytica and Giardia intestinalis [134].

The incidence of enteric pathogens was investigated in 265 Jordanian children

suffering from gastroenteritis using PCR and conventional methods. They detected

enteropathogens in 66.4% of the examined children. A single enteric pathogen was

detected in 50.9% of the children, and multiple pathogens were detected in 15.5%.

The prevalence of enteropathogens identified was as follows: Rotavirus (32.5%),

EPEC (12.8%), EAEC (10.2%), ETEC (5.7%), Shigella spp. (4.9%), Entamoeba

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histolytica (4.9%), Salmonella spp. (4.5%), Campylobacter jejuni/coli (1.5%),

Cryptosporidium spp. (1.5%), EIEC (1.5%), Giardia intestinalis (0.8%) and Y.

enterocolitica (0.4%). No Vibrio cholerae, STEC, microsporidia, adenovirus or

small round viruses were detected [135].

In the USA, the regional variation in the incidence of laboratory-confirmed bacterial

foodborne illnesses was studied. 12,125 cases were identified. The incidence per

100,000 population was highest for Campylobacter (15.7%), followed by Salmonella

(14.4%), and Shigella (7.9%). Lower incidences were reported for E. coli O157

(2.1%), Yersinia (0.4%), Listeria (0.3%) and Vibrio (0.2%). The incidence of

Campylobacter and Salmonella among infants proved particularly high, although

substantial regional variations were observed [136].

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CHAPTER III

MATERIALS AND METHODS

3.1 Materials

3.1.1 Bacterial culture media

• Salmonella-Shigella agar (BioMerieux, France).

• Xylose Lysine Deoxycolate agar (BioMerieux, France).

• Hektoen Enteric agar media (BioMerieux, France).

• Selenite broth base (OXOID, England).

• Campylobacter Blood-Free Selective Agar Base (Modified CCDA-Preston)

(OXOID, England).

• CCDA selective supplement (OXOID, England).

• Yersinia selective agar Base (Difco, USA).

• Yersinia antimicrobic supplement CN (Difco, USA).

• Muller Hinton agar (BioMerieux, France).

• MacConkey agar (BioMerieux, France).

• Buffered peptone water (Difco, USA).

• Phosphate buffer saline 7.4 (Sigma, Germany).

3.1.2 Reagents

• Gram stain (BioMerieux, France).

• Oxidase test (Hy.laboratories Ltd, Occupied Palestine).

• Catalase test (BioMerieux, France).

• Salmonella O antiserum poly A-I & Vi (Difco, USA).

• Salmonella H antiserum poly a-z ((Difco, USA).

• Antiserum Shigella dysenteriae polyvalent A1+A2 (Biorad, France).

• Antiserum Shigella boydii polyvalent C1+C2+C3 (Biorad, France).

• Antiserum Shigella sonnei polyvalent (Biorad, France).

• Antiserum Shigella flexneri polyvalent (Biorad, France).

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• The API-20E test kit (BioMerieux, France).

• The API Campy test kit (BioMerieux, France).

• McFarland standard 0.5.

• Sterilized water ampoule (10 ml).

3.1.3 Supplies

The following supplies were purchased from local suppliers in Gaza strip:

• Glass slides.

• Petri dishes.

• Plastic loop.

• Sterile cotton swab.

• Sterile saline.

• Antibiotic discs (Himedia, India).

• Sterile cup.

• Syringe 5ml.

• Needle.

• CampyGen (microaerophilic environment pack).

3.1.4 Apparatus and equipments

• Incubator 37°C (Jouan, USA).

• Incubator 28°C (Ari J. levy, Occupied Palestine).

• Incubator 42°C (Seleta, Switzerland).

• Anaerobic jar (OXOID, England).

• Microscope (Olympus, USA).

• Safety cabinet (BK1, France).

• Refrigerator (Amcor, Occupied Palestine).

• Autoclave (Tuttnauer, USA).

• Vortex Mixer (Digisystem, Taiwan).

• Balance (Mettler, Switzerland).

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• Computer (LG, China).

• Digital camera (Nokia, Finland).

3.2 Study area

The study was performed in Primary Health Care Clinics (PHCC) at Ministry of

Health (MOH) in Gaza strip. The study was conducted during the period from Jan

2010 to February 2010. Patients were recruited in 6 primary health care centres in

Gaza strip located in Gaza area, Northern area, and Middle area and Khan Yunis

area.

3.2.1 Samples

A total of 132 stool samples were collected from patients with acute diarrhea

(gastroenteritis). Fresh passed stool samples without any preservatives were collected

in sterile containers. Cases are patients attending the PHCC for diarrhea (defined as

the passage of 3 or more loose or liquid stools per day). The age categories of

patients are < 5 and > 5 years old. In addition, data were collected from 132 healthy

controls visited the PHCC at the same time of samples collection from the patients.

Healthy controls had the same characteristics of patients such as (age, gender),

except the fact they don’t suffer from diarrhea within the last three months or at

birth.

3.2.2 Sample transport

All stool samples were transported to the laboratory in the sterile container, and

transported in an icebox immediately after collection. Samples were completely

labelled by the necessary data (date, time of collection, sample type, patient name).

3.2.3 Questionnaire The questionnaire used in this study-included information about age, sex, domestic

animals contact, sources of drinking water, symptoms, including the presence of

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diarrhea….etc (see annex1). Data were collected by interviewing patients, controls or

patient's guardians.

3.2.4 Permissions and ethical consideration

Ethical approval was obtained from the concerned authorities for sample collection,

including Helsinki committee, Doctors of the World (Médecins du Monde - France)

which helped to collect data and financially supported this work. The participating

patient's guardians signed informed consent.

3.2.5 Data analysis

Data was collected, summarized, tabulated and analyzed using Statistical Package for

Social Sciences (SPSS) software. The results were presented through tables, and pie

charts, Chi-square test was used when appropriate.

3.3 Isolation and identification procedures of enteropathogenic bacteria

3.3.1 Salmonella and Shigella

Each stool sample was directly cultured onto Xylose Lysine Deoxycolate agar (XLD)

agar, Salmonella Shigella (SS) agar, Hektoen enteric (HE) agar. Approximately 1 g

of each sample inoculated into 10 ml of Selenite F broth. The tubes and plates were

incubated at 37ºC for 18 to 24 hr. Selective Selenite F broth subcultured onto XLD

agar, SS agar, and HE agar then the plates incubated at 37°C for 18 to 24 hr. The

suspected colonies identified by colony morphology and biochemical characteristics.

Salmonella spp. appears on SS, XLD and HE agar as colorless colonies with black

centre owing to H2S production. Shigella spp. colonies identified on SS agar as

colorless while on XLD and HE agar the colonies appeared as transparent red.

Standard biochemical tests like analytical profile index (API) 20E system and

specific anti sera were used for the confirmation of Salmonella and Shigella [137]

(figure 3.1).

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Stool

samples

Salmonella

and Shigella

Campylobacter

Yersinia

enterocolitia

Culture on Yersinia agar and incubated at 28 ºC for 48

hr.

Aeromonas

hydrophila

Culture on CCDA.

Incubated at 42 ºC for 48

hr

Culture on XLD, SSA,

HE, Selenite F broth

Identified by colony

morphology oxidase test

Cold enrichment

method incubated at 4 ºC for 3 weeks

Identified by colony

morphology Biochemicals Gram stain

Identified by colony

morphology

Biochemical tests like API20E system

Confirmation by

agglutination with antisera

Biochemical tests like API campy system

Inoculated onto Yersinia

agar then identified by morphology

Biochemical tests like API20E system

Culture on XLD, SSA

HE, Selenite F broth

Biochemical tests like API20E system

Figure (3.1): Isolation and identification procedures of enteropathogenic bacteria.

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3.3.2 Campylobacter

Each stool sample was cultured onto (Modified CCDA). The alkaline peptone water

was used as enrichment media in which samples were incubated at 42ºC for 24 hr

and then inoculated onto CCDA. The plates were incubated at 42ºC for 48 hr in

microaerophilic conditions of growth with CampyGen system (5%O2, 10%CO2 and

85%N2). Campylobacter jejuni colony morphology may appear as small, mucoid,

greyish, flat colonies with irregular edges and no hemolytic patterns after 24-48 hr.

They may also appear as round, convex, entire, glistening colonies 1-2 mm in

diameter. Certain strains of C. jejuni may appear lightly pink or tan in color. The

suspected colony of Campylobacter was identified by gram stain and biochemical

tests such as oxidase, catalase tests and API campy system [138,139].

3.3.3 Yersinia enterocolitica

For isolation of Y. enterocolitica, each stool samples was cultured onto Yersinia

selective agar and incubated at 28ºC for 48 hr. The cold enrichment method was used

in which samples incubated at 4ºC for 3 weeks with phosphate buffer saline (PBS)

then inoculated onto Yersinia selective agar and MacConkey agar which are

incubated at 28ºC and 37ºC for 48 hr. The suspected colony growth appear as; red

centre, transparent borderlines and identified by biochemical testes (API20E system)

[95]

3.3.4 Aeromonas hydrophila

Each stool samples was cultured onto XLD agar, SS agar, HE agar and Yersinia

selective agar. Approximately 1 g of each sample inoculated into 10 ml of Selenite F

broth. The tubes and plates were incubated at 37ºC for 18 to 24 hr. Yersinia selective

agar were incubated at 28ºC for 48 hr. Selective Selenite F broth subcultured onto

XLD agar, SS agar, and HE agar then the plates incubated at 37°C for 18 to 24 hr.

The suspected colonies identified by colony morphology and biochemical

characteristics. Colonies from any place resembling Aeromonas spp. were screened

for the production of oxidase. Definitive identification was performed by using

conventional biochemical methods and API20E system [140].

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3.4 Antimicrobial susceptibility for the bacterial isolates

Disk diffusion testing is one of several phenotypic assays, which can be utilized to

determine the antimicrobial resistance profile (antibiogramme) of an organism. Disk

diffusion tests estimate in vitro susceptibility.

Agar plates were inoculated with standardized inoculums of the bacteria and the

appropriate antimicrobial disks were placed on the inoculated agar plate. The disks

used for a disk diffusion assay contain a standardized known amount of an

antimicrobial agent, which diffuses into the agar when in contact with the agar

surface. The plates were incubated at 37oC. During incubation, the antimicrobial

agent diffuses into the agar and inhibits growth of the bacteria, producing a “zone of

inhibition” around the disk [141].

Campylobacter spp. were grown in CCDA agar, at 42ºC, under a microaerophilic

condition (85% N2, 10% CO2, 5% O2), for 48hr. After incubation, all strains were

suspended in PBS pH 7.4 and the density was adjusted to the 0.5 McFarland turbidity

standards and inoculated on Mueller-Hinton agar plates supplemented with 5% of

whole blood. Disks of antimicrobials were then placed onto the plates and were

incubated at 37ºC under a microaerophilic atmosphere, for 48 hr and the inhibition

zones were measured [142].

3.5 Staining and biochemical tests

3.5.1 Gram stain

One small drop of saline was placed on a slide. Colonies of suspected bacteria were

mixed with saline and smeared over the surface of the slide. The smears are allowed

to dry thoroughly. The smears are fixed by passing the slide; smear up, quickly

through the Bunsen flame three times. After cooling, the smears were stained.

Between each staining reagent, the smear is washed under a gently running tap,

excess of water tipped off before the next reagent is added [143].

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3.5.2 Oxidase test

One colony of the test organism is transferred to a filter paper soaked with oxidase

reagent (tetramethyl-p-phenylenediamine dihydrochloride). Appearance of a blue

color within 10 seconds indicates a positive result [143].

3.5.3 Catalase test

A colony is placed at a small spot on a slide. One drop of 3%-H2O2 is added on the

spot with the bacterial material, examined immediately for evolution of gas, which

indicates catalase activity [143].

3.5.4 API 20 E test

The (API) 20E strips (BioMerieux) was used as biochemical system for identification

of gram-negative rod bacteria. The API 20E strip consists of 20 micro tubes

containing dehydrated substrates. These strips were inoculated with bacterial

suspension, which reconstitutes the media. The strips were incubated for 18 to 24 hr

at 37ºC during incubation; metabolism produces changes that are either spontaneous

or revealed by the addition of reagents. The results were recorded and interpreted

according to reading table and the identification was obtained by referring to the

API20E catalogue (BioMerieux, France).

3.5.5 API campy test:

The API Campy strip consists of 20 microtubes containing dehydrated substrates. It

is made up of two parts. The first part of the strip (enzymatic and conventional tests)

is inoculated with a dense suspension, which rehydrates the substrates. During

incubation (in aerobic conditions), metabolism produces color changes that are either

spontaneous or revealed by the addition of reagents. The second part of the strip

(assimilation or inhibition tests) is inoculated with a minimal medium and incubated

in microaerophilic conditions. The bacteria grow if they are capable of utilizing the

corresponding substrate or if they are resistant to the antimicrobial tested.

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The reactions are read according to the reading table and the identification is

obtained by consulting the profile list in the package insert (BioMerieux, France).

3.6 Specific antisera

Confirmation of the results was performed for Salmonella, Shigella isolates by

specific antisera.

3.6.1 Serotyping of Shigella spp.

Serological identification of Shigella begins with the use of polyvalent antisera,

which is used to identify the species (i.e. S. dysenteriae, S. boydii, S. flexneri, or S.

sonnei). If agglutination is observed with polyvalent antisera, the isolates are then

tested with the individual monospecific serum found in the polyvalent antisera [144].

3.6.2 Serotyping of Salmonella spp.

Serotyping of Salmonella strains is carried out by identification of surface antigens

(LPS, O-antigens) and flagella antigens (proteins, H-antigens). Most commonly,

strains of Salmonella express two phases of H- antigens but aphasic, monophasic and

triphasic variants are known [145].

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CHAPTER IV

RESULTS

4.1 Description of study samples

4.1.1 Distribution of the samples in terms of residence area of the study

samples

This investigation is a matched case-control study with prospective data record.

Cases (132) are patients attending the health care centre with diarrhea, which defined

as the passage of 3 or more loose or liquid stools per day. Controls (132) are patients

attending the PHCC for any other cause and who did not experienced diarrhea within

the last 3 months or since birth. Cases and controls are matched for the date of

inclusion, age category, gender and the PHCC. Patients were recruited in 6 PHCC in

Gaza strip located in Gaza area, Northern area, Middle area and Khan Yunis area

(Table 4.1) from 26/1/ 2010 to 17/2/2010. PHCC were selected from different

geographical locations to represent the whole Gaza strip.

Table (4.1): Primary health care centres included in this study

Name location Samples

Number %

Jabalia Martyrs Northern area 25 19%

Al Zaitoun Gaza area 26 20%

Sabha Gaza area 24 18%

Der Al Balah Martyrs Middle area 23 17%

Al Nusairat Martyrs Middle area 9 7%

Bander Khan Yunis Khan Yunis area 25 19%

Total 132 100%

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4.1.2 Sex and age distribution of the study samples

According to the results (Table 4.2) among cases and controls, female constituted

(56%) while males were (44%) (P = 0.549). (75%) of cases and controls with age

group less than 5 years, (25%) more than 5 years, (P = 0.556).

Table (4.2): Distribution of the cases and controls due to sex and age

P value Total n=(264)

Control n=(132)

Case n=(132)

% N0. % N0. % N0.

0.549

44 116 44 58 44 58 Male

Gender 56 148 56 74 56 74 Female

0.556

75 198 75 99 75 99 < 5 years

Age 25 66 25 33 25 33 > 5

years

4.1.3 Socioeconomic status of the study samples

Among cases there was (29.5%) refugee, and (33.3%) among controls is refugee (P =

0.298). It was found that (15.2%) from cases live in houses with < 5 household

members, while (19.7%) from controls live in houses with < 5 household members

(P = 0.209). There were significant differences between diarrhea, and houses with

low rooms number, and number of household workers (P = 0.019, 0.006

respectively), table (4.3).

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Table (4.3): Distribution of the cases and controls due to socioeconomic status

Significance P < 0.05 *

4.2 Isolation and identification of enteropathogenic bacteria

4.2.1 Salmonella and Shigella

All samples were tested for Salmonella and Shigella using colony morphology

(Figure 4.1), biochemical properties (Figure 4.2), and agglutination with specific

antisera (Figure 4.3). Among 12 enteropathogenic bacteria isolated, there were three

Salmonella spp. and two Shigella spp., which are all Shigella boydii.

Figure (4.1): Colony morphology for Salmonella (A: on XLD and HE, B: on SS Agar), and Shigella (C: on HE Agar and SS).

P value 2χ Control Case

% N0. % N0. (n=264)

0.298 33.3 44 29.5 39 yes Refugee

status 66.7 88 70.5 93 No

0.209

19.7 26 15.2 20 <5 Number of household member 80.3 106 84.8 112 > 5

0.019*

6.1 8 12.9 17 < 2 Number of rooms in the house

71.2 94 55.3 73 2-3

22.7 30 31.8 42 > 3

0.006*

81.1 107 66.7 88 ≥ 1 Number of household workers 18.9 25 33.3 44 None

A C B

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Figure (4.2): API 20E results for isolated bacteria A: Salmonella, B: Shigella. Figure (4.3): Agglutination with specific antisera. A: positive for salmonella, B: negative. 4.2.2 Campylobacter Isolation of Campylobacter spp. from stool samples after culturing onto (Modified

CCDA) (Figure 4.4), incubated at 42oC in microerophilic condition (Figure 4.5) and

identified by API campy test (Figure 4.6). Among 12 enteropathogenic bacteria

isolated, there were three Campylobacter spp.: two Campylobacter coli, and one

Campylobacter jejuni.

Figure (4.4): Colony morphology for Campylobacter spp. On (Modified CCDA).

A B

A B

A B

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Figure (4.5): (Modified CCDA) incubated in microerophilic condition at 42oC.

Figure (4.6): Identification of Campylobacter spp. by API campy test. 4.2.3 Yersinia enterocolitica

Isolation of Y. enterocolitica from stool samples after culturing onto Yersinia

selective agar (Figure 4.7) and identified by biochemical testes (API20E system)

(Figure 4.8). It was found that among 12 enteropathogenic bacteria isolated there

were one Yersinia enterocolitica.

Figure (4.7): Colony morphology for Y. enterocolitica on Yersinia selective agar.

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Figure (4.8): Identification of Y. enterocolitica by API 20E.

4.2.4 Aeromonas hydrophila

Isolation of A. hydrophila from stool samples after culturing onto XLD agar, SS

agar, HE agar and Yersinia selective agar (Figure 4.9). In addition, these samples

were identified by biochemical testes (API20E system) (Figure 4.10), among 12

enteropathogenic bacteria isolated, there were three A. hydrophila.

Figure (4.9): Colony morphology for A. hydrophila, A: on XLD., B: on SS agar, C: HE.

Figure (4.10): Identification of A. hydrophila by API 20E.

A

C B

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Isolated enteropathogenic bacteria

Salmonella25.0%

Aeromonas hydrophilia

25.0%

Shigella16.7%

Campylobacter coli/jejuni

25.0%

Yersinia enterocolytica

8.3%

4.2.5 Number and types of isolated enteropathogenic bacteria among the

study samples

Using standard bacteriological culture methods, twelve enteropathogenic bacteria

were detected in twelve cases (9.1%) of the 132 stool samples examined. Figure

(4.11) shows the distribution of enteropathogenic bacteria. Salmonella,

Campylobacter coli/jejuni, and A. hydrophilia were isolated in equal numbers from

samples 3/12 (25% each), Shigella 2/12 (16.7%), and Y. enterocolytica 1/12 (8.3%).

The two Shigella spp. are Shigella boydii.

Figure (4.11): Enteropathogenic bacteria isolated from the samples.

4.2.6 Isolated enteropathogenic bacteria distributed by residence Most of enteropathogenic bacteria 5/12 (41.7%) were isolated from Bander Khan

Yunis, while 2/12 (16.7%) from Jabalia Martyrs, 2/12 (16.7%) from Al Zaitoun, 1/12

(8.3%) from Sabha, Der Al Balah Martyrs, and Al Nusairat Martyrs, respectively as

shown in table (4.4).

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Table (4.4): The isolated enteropathogenic bacteria by residence

Enteropathogen (n=12)

Area* (PHCC) Total 2=132 1

(n=25)

2

(n=26)

3

(n=24)

4

(n=23)

5

(n=9)

6

(n=25)

Salmonella 1 0 0 0 0 2 3

Shigella 0 1 1 0 0 0 2

Yersinia

enterocolytica 0 0 0 0 0 1 1

Campylobacter coli/jejuni 1 1 0 0 1 0 3

Aeromonas hydrophilia 0 0 0 1 0 2 3

Total Positive (%) 16.7% 16.7% 8.3% 8.3% 8.3% 41.7% 100

* Area1: Jabalia Martyrs, area 2: Al Zaitoun, area 3: Sabha, area 4: Der Al Balah

Martyrs, area 5: Al Nusairat Martyrs, area 6: Bander Khan Yunis.

4.2.7 Occurrence of enteropathogenic bacteria among the different age groups From table (4.5), it could be observed that enteropathogenic bacteria were isolated

with a higher frequency from patients belonging to the age group less than 5 years

(66.7%), in comparison to (33.3%) of enteropathogenic bacteria isolated from

patients > 5 years old. There was a decline in prevalence of all pathogens with

increasing age. An association was found among isolated enteropathogenic bacteria

and age groups less than 5 years (P = 0.558) but there was no statistical significance.

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Table (4.5): Enteropathogenic bacteria among the different age groups

Bacteria Age Groups

<5 Years n=99)(

5 Years n=33)(

P value

No. % No. %

0.558

Salmonella 2 1.5 1 0.76

Shigella 2 1.5 0 0

Y. enterocolytica 1 0.76 0 0

Campylobacter Coli/Jejuni 2 1.5 1 0.76

Aeromonas hydrophilia 1 0.76 2 1.5

Total 8 6.1 4 3.0

4.2.8 Distribution of enteropathogenic bacteria according to gender As shown in table (4.6), an equal number of enteropathogenic bacteria were isolated

from both male and female. From male: Salmonella and A. hydrophilia account for

(1.5%), whereas Shigella and Campylobacter coli/jejuni account for (0.76%).

Whoever, for female the percentage was (0.76%) for Salmonella, Shigella, A.

hydrophilia and Y. enterocolytica, and (1.5%) for Campylobacter coli/jejuni, (P =

0.736). Table (4.6): Enteropathogenic bacteria among the gender

bacteria

Gender

P value

Male(n=58) Female(n=74) No. % No. %

0.736

Salmonella 2 1.5 1 0.76

Shigella 1 0.76 1 0.76

Y. enterocolytica 0 0 1 0.76

Campylobacter coli/jejuni

1 0.76 2 1.5

Aeromonas hydrophilia

2 1.5 1 0.76

Total 6 4.5 6 4.5

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4.2.9 Clinical symptoms in relation to presence of isolated bacteria Table (4.7) presents the symptoms in relation to isolated enteropathogenic bacteria in

patients with diarrhea, this table showed that there were no association between

chronic diseases and the presence of enteropathogenic bacteria because no one had

chronic disease among the positive culture cases. In addition, most cases (58.3%) had

mucous in stool, another symptoms present among cases in different rates as fever

(25%), chills (25%), vomiting (50%), bloody diarrhea (16.7%), and loss of weight

(33.3%). Table (4.7): Clinical symptoms in relation to presence of bacterial enteropathogens.

(n=12)

Number of household members reported diarrhea within 10 days before patient’s illness

<1 >1 No. % No. %

10 83.3 2 16.7 Received antibiotics within 4 weeks before the beginning of the diarrhea

yes no No. % No. % 1 8.3 11 91.7

Chronic disease yes no

No. % No. % 0 0 12 100

Fever No Fever Fever

No. % No. % 9 75 3 25

Chills yes no

No. % No. % 3 25 9 75

Vomiting yes no

No. % No. % 6 50 6 50

Bloody diarrhea yes no

No. % No. % 2 16.7 10 83.3

Mucous in stool yes No

No. % No. % 7 58.3 5 41.7

Loss of weight yes No

No. % No. % 4 33.3 8 66.7

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4.2.10 Correlation of the socioeconomic status of the study samples with

isolated enteropathogens

From table (4.8), it could be observed that enteropathogenic bacteria were isolated

with a higher frequency from patients living in a crowded houses, 10/12 (83.3%) of

enteropathogenic bacteria isolated from patients who live in houses with > 5

household members, while (16.7 %) of isolated enteropathogenic bacteria isolated

from patients who live in houses with < 5 household members, and also a higher

frequency of enteropathogenic bacteria were isolated from patients who live in

houses with low rooms numbers, whereas 10/12 (83.3%) of enteropathogenic

bacteria were isolated from patients who live in 2-3 rooms in the house, (16.7%)

from patients who live in > 3 rooms in the house. A statistical significant association

was found between diarrhea and houses with low room's numbers.

Table (4.8): Socioeconomic status of the study samples with isolated enteropathogenic

bacteria

Table (4.8): Socioeconomic distribution of the study population with diarrhe

P value Total Yersinia enterocolytica

Aeromonas hydrophilia Shigella Campylobacter Salmonella (n=132)

0.438

5 3.8%

0 0%

1 0.76%

2 1.5%

1 0.76%

1 0.76%

Yes Refugee status 7

5.3% 1

0.76% 2

1.5% 0

0% 2

1.5% 2

1.5% NO

0.663

2 1.5%

0 0%

0 0%

0 0%

1 0.76%

1 0.76%

<5 Number of household member 10

7.6% 1

0.76% 3

2.3% 2

1.5% 2

1.5% 2

1.5% > 5

0.463

0 0%

0 0%

0 0%

0 0%

0 0%

0 0%

< 2

Number of rooms in the house

10 7.6%

1 0.76%

3 2.3%

1 0.76%

3 2.3%

2 1.5%

2-3

2 1.5%

0 0%

0 0%

1 0.76%

0 0%

1 0.76%

>3

0.216

9 6.8%

1 0.76%

3 2.3%

1 0.76%

1 0.76%

3 2.3%

≥ 1 Number of household workers 3

2.3% 0

0% 0

0% 1

0.76% 2

1.5% 0

0% None

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4.3 Antimicrobial susceptibility profile

4.3.1 Antimicrobial profile of enteropathogenic bacteria isolated from

patients

The antimicrobial profile for isolated enteropathogenic bacteria to various

antimicrobial drugs was determined by the disk diffusion method following the

recommendations of the CLSI. According to table (4.9) the antimicrobial profile of

twelve isolated enteropathogenic bacteria showed high resistance rates for

Campylobacter coli/jejuni (52.4%), followed by Aeromonas hydrophilia (49.2%),

Yersinia enterocolytica (42.9%), Shigella (26.2%) and Salmonella spp. (22.2%).

In addition, the antimicrobial profile of the isolated enteropathogenic bacteria

showed that antibiotics resistance (91.6%) to rifampin, (83.3%) to clindamycin,

(75%) to erythromycin, (66.6%) to cephalexin, (66.6%) to tetracycline, (58.3%) to

amoxicillin, (58.3%) to cephazolin, (58.3%) to trimethoprim/sulfamethoxazole,

(41.6%) to nalidixic acid, (41.6%) to doxycycline, (33.3%) to azerotenam, (33.3%)

to pipracillin, (33.3%) to cefuroxime, (16.6%) to cloramphinicol, (16.6%) to

cefatoxime, (16.6%) to cefatazidim, (8.3%) to ofloxacin, (8.3%) to ceftriaxone,

(8.3%) to ciprofloxacin and (38.6%) to all antibiotics. All isolates were sensitive to

gentamicin, and amikacin.

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Table (4.9): Antimicrobial susceptibility of isolated enteropathogenic bacteria

Amoxicillin (AM), Pipracillin (PC), Erythromycin (E), Clindamycin (CD), Cephalexin (CP), Cefuroxime (CU), Cefatoxime (CE), Cefatazidim (CAZ), Ceftriaxone (CI),

Cephazolin (CZ), Amikacin (AK), Gentamicin (GM), Tetracycline (TE), Doxycycline (DO), Cloramphinicol (C), Trimethoprim/sulfamethoxazole (SXT), Nalidixic Acid (NA),

Ciprofloxacin (CIP), Azerotenam (AT), Rifampin (RA), Ofloxacin (OFX).

Enteropathogenic

bacteria (n=12)

Antibiotics

Percentage

of resistance

%

total AM\ PC E CD CP CU CE CAZ CI CZ AK GM TE DO C SXT NA CIP AT RA OFX

Salmonella spp. S S R R I I S S S R S S S S S S S S S R S 19

22.2

Salmonella spp. S S R R I S S S S I S S R R S S R S S R S 28.6

Salmonella spp. S S R R R S S S S S S S S S S S S S S R S 19

Shigella spp.

R S S R I I S S S R S S R S S R R S S S S 28.6

26.2

Shigella spp. S S R R R S S I S R S S I S S S S S S R S 23.8

Campylobacter coli/jejuni

R R R R R R R I I R S S R R R R S S R R S 66.7

52.4

Campylobacter coli/jejuni

R R R R R I S S S R S S R R S R S S S R S 47.6

Campylobacter coli/jejuni

I R S S S R S S S S S S R S S R R R R R R 42.9

Aeromonas hydrophilia

R S I R R S S I S I S S S S S S S S S R S 19

49.2 Aeromonas

hydrophilia R R R R R R I R I R S S R R S R S S R R S

61.9

Aeromonas hydrophilia

R S R R R R R R R R S S R S I R R S R R S 66.6

Yersinia enterocolytica

R S R S R I S S S I S S R R R R R S S R S 42.9

Percentage of resistance

58.3 33.3 75 83.3 66.6 33.3 16.6 16.6 8.3 58.3 0 0 66.6 41.6 16.6 58.3 41.6 8.3 33.3 91.6 8.3 38.6

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4.3.2 Prescription of antibiotics to the diarrheal patients

About (30.3%) of the patients used antibiotics, trimethoprim/sulfamethoxazole was

the most antibiotic taken (25%), while amoxicillin (3%), cloxacillin (1.5%),

ampicillin were less prescribed by treating physician (0.8%), and unknown (0.8%).

The percentage of patients who received antibiotic within 4 weeks before the

beginning of the diarrhea was (27.3%) (Table 4.10).

Table (4.10): Prescription of antibiotics to the diarrheal patients

Diarrhea (n=132)

no yes

Antibiotic within 4 weeks before the beginning of the diarrhoea

. % No. % No.

72.7 96 27.3 36

% No. % No. Antibiotic therapy for diarrhea 69.7 92 30.3 40

% No.

Trimethoprim/sulfamethoxazole 25 33

3 4 Amoxicillin

0.8 1 Ampicillin

1.5 2 Cloxacillin

0.8 1 Unknown

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4.4 Correlation of socioeconomic factors with diarrhea

In this study, we collected information from cases and controls about socioeconomic

factors. In this section, we will correlate these socioeconomic factors with diarrhea in

Gaza strip.

4.4.1 Correlation between diarrhea and domestic animals in the house

As shown in table (4.11), there are statistically significant differences between the

presence of poultry and pigeon in the houses and diarrhea because there are poultry

(26.5%) and pigeon (19.7%) in the cases house while there is poultry (11.4%), and

pigeon (8.3%) in the controls group.

On the other hand there are (4.5%), (10.6%), (8.3%) of rabbit, sheep, donkey in the

cases group respectively while there are (9.1%), (5.3%), (9.1%) of rabbit, sheep,

donkey in the in the controls group respectively. This correlation is statistically not

significant.

Table (4.11): Domestic animals in the house and diarrhea

*Significance P < 0.05

P value Control(n=132) Case (n=132)

(n=264) % N0. % N0.

0.001*

11.4 15 26.5 35 Yes Poultry

88.6 117 73.5 97 No

0.111

9.1 12 4.5 6 Yes Rabbit

90.9 120 95.5 126 No

0.086

5.3 7 10.6 14 Yes Sheep

94.7 125 89.4 118 No

0.006* 8.3 11 19.7 26 Yes Pigeon

91.7 121 80.3 106 No

0.500

9.1 12 8.3 11 Yes Donkey 90.9 120 91.7 121 No

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4.4.2 Correlation between diarrhea and some furniture in the houses

As shown in table (4.12) there are association between diarrhea and the presence or

absence of some furniture in the houses, for example there are statistically significant

differences between diarrhea and presence of washing machine, cooking utensils in

the houses because there are (80.3%) of cases had washing machine and (83.3%) of

cases had cooking utensils, while there were (90.9%) of controls had washing

machine, (91.7%) of controls had cooking utensils, in the other hand there are no

statistically significant differences between the presence of fridge, latrine in the

houses and diarrhea.

Table (4.12): Correlation between diarrhea and some furniture in the houses.

P value

Control(n=132) Case(n=132) (n=264)

% N0. % N0.

0.065

87.9 116 80.3 106 Yes Fridge

12.1 16 19.7 26 No

0.011*

90.9 120 80.3 106 Yes Washing machine 9.1 12 19.7 26 No

0.031*

91.7 121 83.3 110 Yes Cooking

utensils 8.3 11 16.7 22 No

0.361

97.7 129 96.2 127 Yes Latrine in the house 2.3 3 3.8 5 No

Significance P < 0.05 *

4.4.3 Correlation between diarrhea and current water access in the houses

There are statistically significant differences between the current water access in the

houses and diarrhea because the current water access in the houses of the controls is

better than in the houses of cases as shown in table (4.13): (Public water access at

home, water from private provider, roof Jerrican, filtered water home: (85.6%),

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(62.9%), (88.6%), (3.8%) in cases and (94.7%), (66.7%), (92.4%), (6.1%) in controls

respectively).

Table (4.13): Current water access in the house

P value Control(n=132) Case(n=132)

(n=264) % N0. % N0.

0.011*

94.7 125 85.6 113 Yes Public water

access at home

5.3 7 14.4 19 No

0.303

66.7 88 62.9 83 Yes Water

from private provider

33.3 44 37.1 49 No

0.200

92.4 122 88.6 117 Yes Roof Jerrican 7.6 10 11.4 15 No

0.286

6.1 8 3.8 5 Yes Filtered water at

home 93.9 124 96.2 127 No

Significance P < 0.05 *

4.4.4 Correlation between diarrhea and drinking water in the house

As shown in table (4.14) tap water in Gaza strip may be contaminated because the

percent of cases drinking tap water are more frequent than those in controls (11.4%

in cases, 9.8% in controls). In addition, there is statistically significant correlation

between using domestic wells water for drinking purposes and diarrhea.

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Table (4.14): Drinking water in the house

P value Control(n=132) Case(n=132)

(n=264) % N0. % N0.

0.421 9.8 13 11.4 15

Yes

Tap water

90.2 119 88.6 117 No

0.500 89.4 118 88.6 117

Yes

Filtered water

10.6 14 11.4 15 No

0.142 6.8 9 11.4 15

Yes Boiled

water 93.2 123 88.6 117

No

0.286 13.6 18 10.6 14

Yes

Bottle water

86.4 114 89.4 118 No

0.042* 2.3 3 7.6 10

Yes

Domestic wells

97.7 129 92.4 122 No

0.247 25.8 34 30.3 40

Yes Water access changes

74.2 98 69.7 92 No

Significance P < 0.05 *

4.4.5 Correlation between diarrhea and sewage disposal

The use of barrels and cesspool in the house increased diarrhea as shown in table

(4.15). Cases who used barrel, and cesspool are (11.4%), (13.6%), while controls

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(8.3%), (11.4%) respectively. Whilst using sewers improve situation of diarrhea and

diminish diarrheal cases (78% using sewers in cases, 80.3% in controls).

Table (4.15): Sewage disposal and diarrhea

P value Control(n=132) Case(n=132)

Swage Disposal (n=264) % N0. % N0.

0.268 8.3 11 11.4 15 Yes

Barrel 91.7 121 88.6 117 No

0.355 11.4 15 13.6 18 Yes

Cesspool 88.6 117 86.4 114 No

0.381 80.3 106 78 103 Yes

Sewers 19.7 26 22 29 No

0.300 15.9 21 12.9 17 Yes Sewage

Disposal Changes 84.1 111 87.1 115

No

4.5 Clinical signs and symptoms of diarrheal patients As shown in table (4.16), certain signs and symptoms during diarrhea may be

dominant such as mucous in stool (65.2%), or present at low percentage such as

chills (31.1%), bloody diarrhea (14.4%). Another sign and symptoms were present at

high percentage are vomiting (50%), loss of weight (50%), and fever (40.9%). Only

(5.3%) from cases had chronic diseases.

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Table (4.16): Clinical signs and symptoms of diarrheal patients Sign and symptoms Percentage (n=132)

Number of household members reported

diarrhea within 10 days before patient’s illness

<1 >1 No. % No. % 100 75.8 32 24.2

Chronic disease yes no

No. % No. % 7 5.3 125 94.7

Fever No Fever Fever

No. % No. % 78 59.1 54 40.9

Chills yes no

No. % No. % 41 31.1 91 68.9

Vomiting yes no

No. % No. % 66 50 66 50

Bloody diarrhea yes no

No. % No. % 19 14.4 113 85.6

Mucous in stool yes No

No. % No. % 86 65.2 46 34.8

Lost of weight yes No

No. % No. % 66 50 66 50

4.6 Management and follow-up of diarrheal patients During management and follow-up of diarrheal patients after one week from

admission time, its clear that most of cases (78.8%) were recovered, diarrhea

persisted in (19.7%), referred to hospital (0.8%), new attendance at the same PHCC

(0.8%), death due to diarrhea (0.0%), lost to follow-up (1.5%), and positive culture

(9.1%) .

Dehydration is a very serious consequence of diarrhea, so (48.5%) of cases obtained

rehydration with oral rehydration salts (ORS) or equivalent, and (2.3%) from cases

needs to be referred to the hospitals (table 4.17).

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Table (4.17): Management and follow-up of diarrheal patients (n=132)

Recovered

yes No unknown

No. % No. % No. % 104 78.8 26 19.7 2 1.5

Persistence of

diarrhea

yes No unknown No. % No. % No. %

26 19.7 104 78.8 2 1.5

Referred to hospital

yes No unknown

No. % No.

% No. %

1 0.8 129 97.7 2 1.5

New attendance at the

same PHCC

yes No unknown

No. % No.

% No. %

1 0.8 129 97.7 2 1.5

Death due to diarrhea

yes No unknown

No. % No.

% No. %

0 0 130 98.5 2 1.5

Lost case to follow-up

yes No No. % No. % 2 1.5 130 98.5

Positive culture

yes No No. % No. % 12 9.1 120 90.9

Rehydration with ORS yes No

No. % No. % 64 48.5 68 51.5

Needs to be referred to the hospital

yes No No. % No. % 3 2.3 129 97.7

ORS: Oral rehydration salts. Unknown: Cases lost and did not return to PHCC for follow-up.

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CHAPTER V

DISCUSSION

Each year, an estimated 2.5 billion cases of diarrhea occurs among children under

five years of age, and estimates suggest that overall incidence has remained relatively

stable over the past two decades [146]. Diarrheal diseases represent the most

infectious disease in Gaza strip during 2009 (55.6 %) [147]. Infectious diarrhea

affects mainly children who are at risk of complications, especially when they suffer

from malnutrition, which is common in Palestinian children [148].

Poor sanitation and restriction to water access may favour communicable diseases,

especially infectious diarrhea, which is one the leading causes of morbidity in the

Gaza strip [149], and is one of the more frequent reasons for PHCC attendance in

addition to respiratory tract infections [150].

This study aims to analyze enteropathogenic bacteria, antibiotic resistance for

isolated enteropathogenic bacteria and associated-risk factors related to diarrhea.

5.1 Children age less than 5 years old are more susceptible to

infection

Acute diarrhea is one of the most common illnesses in children and a common reason

for doctor's visits. Often it can occur in several members of a family or a classroom

at the same time. Diarrhea is a common gastrointestinal disease occurring as acute or

chronic forms especially in children. It could be associated with growth retardation

or even death in children [151].

According to our results, children less than 5 years old are more susceptible to

infectious diarrhea because 75% of diarrheal patients belong to this age, and

enteropathogenic bacteria were isolated with a higher frequency (66.7%) from

patients belonging to this age group. Many studies are in agreement with our results

that children age less than 5 years old are more susceptible to infectious diarrhea

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[152]. Worldwide, diarrheal diseases are a leading cause of pediatric morbidity and

mortality, with 1.5–2.5 million deaths estimated to occur annually among children

aged < 5 years [6,153,154].

5.2 Diarrhea and enteropathogenic bacteria is more frequent in

crowded houses with low rooms number

High morbidity and mortality from infectious diarrhea in Gaza strip, and outbreaks of

bloody diarrhea are closely related to poverty, malnutrition, and lack of access to

care. Lack of safe drinking water and proper nutrition, grossly inadequate sanitation

and hygiene, crowding with lack of household ventilation and personal indoor air

pollution are all-important living conditions that promote diarrheal diseases in Gaza

strip. In addition, refugees and internally displaced persons are at especially high risk

[40,155]. The risk of transmission of endemic communicable diseases, such as acute

respiratory infections and diarrheal diseases is increased in displaced populations due

to associated crowding, inadequate, unsafe drinking water, and sanitation and poor

access to health care [156,157].

According to this study, diarrhea is more frequent in crowded houses, (84.8%) from

cases living in houses with > 5 household members, and most of cases (55.3%) live

in houses with 2-3 rooms in the house. Moreover more enteropathogenic bacteria

were isolated from crowded houses, (83.3%) of enteropathogenic bacteria isolated

from cases live in house with < 5 household member, (83.3%) isolated from cases

live in houses with 2-3 rooms. These results suggest that there were significant

differences between diarrhea, and houses with low room's number.

5.3 Diarrhea is more frequent in houses rearing poultry, sheeps, and

pigeons

Varieties of domestic animals are capable of transmitting disease to people including:

poultry, and pigeons. According to the results of this study, there was statistically

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significant correlation between the presence of poultry and pigeon in the houses and

diarrhea. This is probably because those who live in rural areas and have regular

contact with livestock are at greater risk of Campylobacter infection (and other

enteropathogens) [158-161]. Animals can actually pass Salmonella on to other

animals, as well as to people. Cattle, chicken, and shellfish are not the only food

animals that can cause a person to become infected with Salmonella. Other animals

that can harbor Salmonella include turkeys, sheep, and swine [26]. In addition,

Salmonella, Campylobacter coli/jejuni, A. hydrophilia, Y. enterocolytica can be

isolated and transmitted from a variety of domestic animals [26,27,162,163].

Meanwhile, there was no statistically significant correlation between diarrhea and

presence of rabbit, sheep and donkeys in the houses. This may be due to these

animals are normally present outside the houses while poultry and pigeon are

normally present inside the houses and close contact is anticipated.

5.4 Improvement in sewage disposal is vital in the reduction of

diarrheal diseases

During the 1800's, the inefficient sewage system led to the outspread of infectious

diseases such as cholera, typhoid, yellow fever, malaria and various other mosquito

diseases [164]. The underdeveloped wastewater storage and treatment facilities and

unchecked sewage flow in the Gaza strip further contaminates groundwater [165].

In accordance with this study findings, access to proper sewage disposal decreased

diarrheal infections (78%) uses sewers among cases while (80.3%) uses sewers,

among controls). While using Barrel, and cesspool in the house increased the

diarrheal infections because cases who used Barrel, and cesspool were (11.4%),

(13.6%), while controls (8.3%), (11.4%) respectively. These results may indicate that

the water-related health problems are wide spread in the Gaza strip, and so United

Nations Relief and Works Agency (UNRWA) reports that among the infectious

diseases affecting the refugee population in the Gaza strip, those that have the

highest rates of occurrence are those directly related to inadequate supplies of safe

water and poor sanitation: watery diarrhea, acute bloody diarrhea and viral hepatitis

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[166]. This contamination of water may be caused by the Israeli military operations

of December 2008-January 2009, which reported to have damaged 2445 meters of

sewage pipes, the sewage network, including sewage treatment plants and pumping

stations, suffered damage at four sites. The electricity network was also damaged,

and the resulting power cuts affected water supplies, wastewater pumping, and

treatment systems [167].

Our findings are in agreement with other studies conducted in developing countries.

One of these studies was conducted to estimate the prevalence and associated factors

for acute respiratory conditions and diarrhea among children under the age of five

years in Iraq in 2000, they found that history of diarrhea and acute respiratory

infections were negatively associated with lower socio-economic status, adequate

disposal of children's stool and dirty water, but the results were inconsistent in terms

of access to potable water and sanitation facilities possibly due to non-functioning of

water and sewage plants after the war [168]. Another study in Nicaragua showed that

simple, low cost interventions that improve water and latrine infrastructure might

reduce the prevalence of diarrheal disease in the isolated regions of Nicaragua and

Central America. Many other studies suggested that inadequate sewage disposal

could be cause infectious diarrhea [169-172]. These studies in addition to our study

showed that improper treatment of sewage in Gaza strip lead to contaminate drinking

water and contribute to the transmission of diseases especially diarrhea and acute

gastroenteritis.

5.5 Correlation between water access, drinking water and diarrhea

Drinking water is water used for domestic purposes, drinking, cooking and personal

hygiene. Access to drinking water means that the source is less than 1 kilometre

away from its place of use and that it is possible to reliably obtain at least 20 litters

per member of a household per day. Safe drinking water is water with microbial,

chemical and physical characteristics that meet WHO guidelines or national

standards on drinking water quality. Access to safe drinking water is the proportion

of people using improved drinking water sources: household connection, public

standpipe, borehole, protected dug well, protected spring, rainwater. About 2.6

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billion people (half the developing world) lack even a simple (improved) latrine and

1.1 billion people have no access to any type of improved drinking source of water.

As a direct consequence, 1.6 million people die every year from diarrheal diseases

attributable to lack of access to safe drinking water and basic sanitation and 90% of

these are children under 5, mostly in developing countries [173].

For decades, the Gaza strip has been plagued by extremely poor water quality. The

sole source of fresh water for the Gaza strip is the Coastal Aquifer. This water source

is deteriorating and contaminated. With more water, being abstracted (pumped) from

the aquifer annually than natural recharge rates, seawater and surrounding saline

aquifers intrude into this fresh water source causing salination [165].

According to our study there was statistically significant correlation between the

current water access in the houses and diarrhea because the current water access in

the houses of the controls is better than in the house of cases as shown in table (4.13).

In addition, tap water in Gaza strip may be contaminated because the percent of cases

drinking tap water is higher than those in the controls (11.4% in cases, 9.8% in

controls). There is a statistically significant correlation between using domestic wells

and diarrhea. It worthy to mention that the Israeli military operations of December

2008 – January 2009 are reported to have damaged 11 public wells and four

reservoirs as well as 19920 meters of water pipes . A total of 5200 households lost

their roof water tanks, and another 2355 household water tanks were damaged.

Nearly 10% of the population of the Gaza strip (over 100,000 people) had no proper

water supply in February 2009. Three months later, 32,000 people still had no proper

water supply [167].

This means the possibility of contamination of tap water and wells in Gaza strip,

which forced the United Nations (UN) recommending an immediate cessation of use

of domestic wells to preserve both the health of the aquifer and the health of those,

that depend on water from it. Poor water quality in Gaza leads to serious health

concerns, with vulnerable groups such as children suffering most [165]. A study in

Nigeria showed that there is association between domestic water sourcing practice

and the risk of developing diarrhea. It is therefore recommended that high premium

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be placed on improving access to water and improved household hygiene as a way of

helping to curb diarrhea [174]. Endemic dysentery is associated with faecal

contamination of water sources [175]. In 2003 in Parbatia, Orissa, Eastern India, an

outbreak of cholerae associated with an unprotected contaminated well [176].

Researchers in Kenyan showed that diarrhea risk was higher among shallow well

users. Chlorinating stored water, latrines, and rainwater use all decreased diarrhea

risk; combined interventions may have increased health impact [177].

5.6 Sign and symptoms of diarrheal patients

Severity of sign and symptoms of diarrheal patients varies from patient to patient

depending on the cause and other factors [178]. Diarrhea may be accompanied by

mucous, chills, vomiting, and fever and lost of weight or an urgent need to use the

bathroom. Most studies revealed that high number of patients had these sign and

symptoms, one of them showed that 76.1% presented with acute watery diarrhea,

(20%) with loose stool, (3.3%) with bloody diarrhea and 0.6% cases with mucoid

diarrhea, vomiting was a predominant clinical feature in 77.7% cases and 35%

suffered from abdominal pain [121]. In another study, Cleary showed that bacterial

gastroenteritis (STEC) usually characterised by the presence of bloody diarrhea,

mucous in the stools and a high fever [179]. Most studies revealed that high number

of patients with gastroenteritis had high fever during diarrhea. In a study, 196

children's assessed between March 1994 and June 1996 at the pediatric emergency

room of the Teaching Hospital, Universidade de Sao Paulo, the results showed the

presence of bloody stools was reported in 11%. The second most frequent symptom

was vomiting, reported in 78.6%. Of whom, 28.6% had three or more episodes in the

previous 24 hours, fever was measured or presumed by guardians in (59%) [128].

In the present study mucous diarrhea were predominant (65.2%), followed by

vomiting and loss of weight (50%), fever (40.9%), chills (31.1%), bloody diarrhea

(14.4%). This study supports the conclusions from other studies that bacterial

enteropathogens induce a clinical illness characterized by fever, mucous diarrhea,

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chills, vomiting, bloody diarrhea, loss of weight, or various combinations of these

symptoms [128,178,179].

In this study when comparing sign and symptoms with isolated enteropathogenic

bacteria, mucoid diarrhea also were predominant (58.3%), fever (16.6%), chills

(25%), vomiting (50%), bloody diarrhea (16.6%), lost of weight (33.3%).

Table 5.1 Clinical features of infection with selected diarrheal pathogens [178].

Pathogen

Yersinia Campylobacter Salmonella Shigella Clinical Feature

O O O Vomiting and nausea

O O O O Bloody stool

Key: common: O = occurs

5.7 Follow-up and convalescence of diarrheal patients

Bacterial gastroenteritis is usually self-limited in the otherwise healthy child. Clinical

recovery is frequently achieved within a few days and excretion of the causative

organisms continues for a relatively short duration, usually not exceeding a few

weeks. Most bacterial gastroenteritis do not require or benefit from antibiotic

treatment [180,181], this in agreement to our study which showed most of cases

(78.8%) recovered after one week of admission time, but (19.7%) have persistence of

diarrhea, (0.8%) referred to hospital, and (0.8%) admitted to the same PHCC. No one

of all dirrahael patients included in this study died from this disease.

In most cases of diarrhea, replacing lost fluid to prevent dehydration is the only

treatment necessary. Medicines that stop diarrhea may be helpful, but they are not

recommended for people whose diarrhea is caused by a bacterial infection. If the

patient stops the diarrhea before having purged the bacteria, he will trap the organism

in the intestines and prolong the problem. Rather, doctors usually prescribe

antibiotics as a first-line treatment [182]. If episodes of diarrhea lasting for less than

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14 days are defined as acute, episodes lasting for more than 14 days are defined as

persistent [183]. Some pathogens, such as Cryptosporidium, Giardia lamblia and

(EAEC) are thought to be associated with persistent or acute diarrhea in some

locations [184]. Most cases of acute infectious diarrhea caused by bacterial enteric

pathogens are self-limiting and recovered within one week [185,186].

5.8 Prevalence of bacterial enteropathogens

According to our study, 12(9.1%) enteropathogenic bacteria were isolated from (132)

dirrahael patients. Salmonella, Campylobacter coli/jejuni, and A. hydrophilia were

isolated in equal numbers from samples 3/12(25% each), Shigella 2/12(16.7%), and

Y. enterocolytica 1/12(8.3%). The two Shigella spp. are Shigella boydii. Low number

of isolated enteropathogenic bacteria may be due to the presence of other causes of

diarrhea such as other enteropathogenic bacteria, parasites and viruses.

This finding is nearly congruent with other local study conducted by Abu Elamreen

et al., (2007) where they reported that (10%) of their samples had enteropathogenic

bacteria which screened by conventional culture method [13]. Other studies reported

higher percentage of enteropathogenic bacteria (17%) in Egypt [117], (40%) in

Jordan [119]. Meanwhile our finding showed that rate of detection of

enteropathogenic bacteria is higher than in a local study in El-Naser Hospital carried

out from 1999 to 2006, which showed that the isolation frequency was (2.6%) for

Salmonella and Shigella, in addition detection rate of Salmonella spp. and Shigella

spp. in Palestine are very low (about 3 cases of Shigella spp and 2 cases of

Salmonella spp. in 2009) [116].

5.9 Distribution of isolated enteropathogenic bacteria by residence

The Gaza coastal line is 42 km long, between 6 and 12 km wide and covers an area

of 365 km2. An estimated 1.5 millions people currently live in Gaza strip. Gaza strip

is composed of five Governorates: Northern, Gaza, Middle, Khan Yunis, and Rafah

Governorate.

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According to our study, the isolation of enteropathogenic bacteria were as follows;

5/12 (41.7%) were isolated from Khan Yunis area, 2/12 (16.7%) from Northern

Gaza, 3/12 (25%) from Gaza zone, 2/12 (16.7%) from Mid zone. It is clear that high

number of enteropathogenic bacteria was isolated from Khan Yunis area; this may be

caused by contaminated drinking water. In fact Khan Yunis area lack wastewater

treatment plant (WWTP). This may cause contamination of groundwater and thus

drinking water because groundwater is the only significant source of water in the

Gaza strip [187,188]. On Tuesday 15 December 2009, Al Mezan Centre for Human

Rights published a report on 'Environment Pollution and Sanitation Problems in

Khan Younis'. The report presents the serious water health and environmental

problems faced by the quarter million people living in Khan Younis city due to the

exacerbating sanitation situation. The report calls for urgent actions to deal with

these problems, which affect a wide array of human rights in Khan Younis, and

threaten water and health in the entire southern Gaza strip. This has contributed to

the deterioration of the underground aquifer, which has reached a critical stage

during 2009. In a more recent development, the report states, two sewage pools were

dug in Al Mawasi area; an agricultural area along Khan Younis beach, where the

south Gaza aquifer is located. The lack of sanitation services pushed many people to

connect their sewage to them. They have become a serious threat to the underground

water and deprived the residents of the area from potable water [189].

Also according to the results 2/3(66.7%) of A. hydrophila were isolated from Khan

Yunis area, this is may be due the wide distribution of A. hydrophila in the

environment in both fresh and salt water, sewage and soil [190-192]. They are

present in high numbers in sewage before and after treatment, thus they have been

proposed as an indicator of sewage-contaminated surface water. Aeromonas spp.

may colonize drinking water distribution systems and produce biofilms that resist

disinfection [193-195].

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5.10 Antimicrobial resistance for isolated enteropathogenic bacteria

Antibiotics were not usually prescribed for gastroenteritis. In view of the high

prevalence of gastroenteritis, this still means that significant amounts of antibiotics

are being prescribed for what remains a self-limiting illness.

According to the present study, 30.3% of cases used antibiotics for diarrhea before

culture result were finished. Trimethoprim/sulfamethoxazole was the most prescribed

antibiotic (25%), in spite of the resistance rate of trimethoprim/sulfamethoxazole was

high (58.3%). These results indicate that there are many physicians prescribing

antibiotics without waiting culture result or they do not request stool culture at all.

The recognition that antibiotic resistance is caused in part by excessive antibiotic

prescribing has prompted calls for reform [196-199]. Yet the optimal methods for

addressing antibiotic resistance problem remain obscure. Because such reform is

likely to require fundamental changes in physicians behaviour [200-203]. A better

understanding of physician's perceptions of antibiotic resistance is essential. In

general, physicians are likely to alter their practice patterns only when their

knowledge, beliefs, attitudes, and skills are aligned with the ends (a reduction in

antibiotic resistance) and the means to achieve them [2].

The last decade showed an alarming increase in antibiotic resistance in infections,

with more than 13 million deaths per year from infections. Inappropriate prescription

of antibiotics prompted resistance and increased infectious disease mortality in

developed countries. Aging populations, changes in behaviour and a decline in the

development of new antibiotics exacerbated a deteriorating situation [2].

In this study multidrug resistance was common among Campylobacter coli/jejuni

which is resistant to (52.4%) of antibiotics, followed by Aeromonas hydrophilia

(49.2%), Y. enterocolytica (42.9%), Shigella (26.2%), and Salmonella (22.2%).

In addition antimicrobial susceptibility testing of isolated enteropathogenic bacteria

showed high resistance rate for rifampin, clindamycin, erythromycin, cephalexin,

tetracycline, cephazolin, amoxicillin, and trimethoprim/sulfamethoxazole. In the

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other hand, some of isolated enteropathogenic bacteria were completely sensitive to

antibiotics such as gentamicin, and amikacin.

Study in India showed that resistance to antimicrobial agents was common among all

pathogens. Among Shigella an overall resistance of (63.6%), (58.1%) and (16.3%)

was observed for nalidixic acid, trimethoprim/sulfamethoxazole and furazolidone

respectively. Seven isolates of Shigella (12.7%) were resistant to ciprofloxacin,

(18.5%) of non-typhoidal isolates were resistant to ciprofloxacin [24]. In another

study in Yemen, more than two-thirds of the Salmonella isolates were resistant to

nalidixic acid, chloramphenicol, trimethoprim/sulfamethoxazole, gentamicin, and

amoxicillin, while (42%) were resistant to cefotaxime. Most of the Shigella isolates

were susceptible to nalidixic acid and cefotaxime, and resistant to the other

antibiotics. All the tested EPEC isolates were resistant to amoxicillin, (83%) were

resistant to trimethoprim/sulfamethoxazole, (62%) to chloramphenicol, and (54%) to

gentamicin, while only (16%) and (6%) were resistant to nalidixic acid and

cefotaxime, respectively [130]. Another study in Salvador, Bahia, Brazil showed that

Shigella spp. presented a very high resistance rate to trimethoprim-sulfamethoxazole

(90.1%) and low resistance to ampicillin (22.0%), while Salmonella presented very

low resistance rates to all drugs tested. These data are useful for practitioners and

they reinforce the need for continuous microbiological surveillance [125].

Prevalence of resistance traits in gram-negative bowel bacteria may be due to misuse

of antibiotics in the health care system, which explains the occurrence of invasive

infections with multiresistant bacilli. These facts are being carefully examined,

leading to epidemiologic and molecular studies that are important in the laboratories,

regarding resistance mechanisms of gram-negative bacilli [131].

5.11 Dehydration caused by diarrhea

Worldwide, 12% of deaths among children less than five years of age are due to

diarrhea [204]. Almost 50% of these deaths are due to dehydration and most involve

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children less than one year of age [205]. Dehydration may be mild or severe (figure

5.1) [178].

Using ORS solutions began in the 1970s as an effective and inexpensive method of

treating mild to moderate dehydration [206]. Despite the success of oral rehydration

therapy (ORT), its proven efficacy [207] and recommendations for use by various

organizations [206,208], studies show that ORT continues to be underused globally

[209], and specifically by physicians in high-income countries [210-213]. A recent

report showed that ORT is being delivered to only (20%) of the world's children who

could benefit and that widespread use could prevent (15%) of deaths among children

under five years [210]. In our study the results showed that ORS is being delivered to

(45.8%) of patients with diarrhea in Gaza strip. This results better than the world's

static's, may be due to abundant of ORS in all PHCC; Government or UNRWA.

Postulated reasons for underuse in the world include the fear of inducing iatrogenic

hypernatremia, time requirements, questionable efficacy in moderate dehydration,

and parental preference [19].

Figure (5.1) Levels of dehydration in children with acute diarrhea [178].

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5.12 Relation of the weather with isolation rate of enteropathogenic

bacteria

The current study was done during the winter season, while enteropathogenic

bacteria are more common in warmer and wetter months, for this reason may be low

number of enteropathogenic bacteria were isolated from the cases. Many studies

suggest that enteropathogenic bacteria are more common in warmer seasons

especially in developing or tropical regions [214]. The prevalence of bacteria in

gastroenteritis is more frequent than Rotavirus in warmer weather. There is a marked

seasonality with sporadic rates of enteropathogenic (Salmonella, Campylobacter,

Shigella) in the United States: the highest rates of infection increase during the late

spring and peak in June, July, or August. The rise in the number of cases during the

summer may be due to higher levels of poultry contamination during warmer

weather, and/or to summer food-consumption patterns, including barbecuing and

eating outdoors, which may result in food that is undercooked or cross-contaminated

[215,216]. A study in Australia showed that the total effect over the preceding week

indicated a relative increase from baseline in the probability of gastroenteritis of

(2.48%) for each degree rise (°C) over that period [217].

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CHAPTER VI

CONCLUSIONS AND RECOMMENDATIONS

6.1 Conclusions

The present study focused on the isolation of enteropathogenic bacteria from stool

samples, estimation of its antibiotic resistance and associated-risk factors related to

diarrhea in the Gaza strip. From this study the following conclusions were drawn:

1. Children younger than 5 years old are more susceptible to infectious diarrhea.

2. A higher frequency (66.7%) of enteropathogenic bacteria was isolated from

patients belonging to the age group younger than 5 years.

3. Diarrhea is more frequent in crowded houses with low room's number,

(84.8%) from cases living in houses with > 5 household members.

4. High percentages of enteropathogenic bacteria (83.3%) were isolated from

crowded houses with > 5 household member.

5. A higher frequency of enteropathogenic bacteria were isolated from patients

living in houses with low rooms numbers, where 10/12(83.3%) of

enteropathogenic bacteria isolated from population who live in 2-3 rooms in

the houses.

6. Diarrhea is more frequent among peoples living in houses rearing poultry,

and pigeons. Statistically significant correlation between the presence of

poultry and pigeon in the houses and diarrhea was shown.

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7. Access to proper sewage disposal decreases diarrheal infections, (78%) uses

sewers in cases, and (80.3%) uses sewers in controls.

8. There are statistically significant differences between the current water access

in the houses, using domestic wells and diarrhea.

9. Mucous diarrhea was predominant (65.2%), followed by vomiting and loss of

weight (50%), fever (40.9%), chills (31.1%) and bloody diarrhea (14.4%).

10. Most cases (78.8%) recovered after one week of admission time, but (19.7%)

have persistent of diarrhea, (0.8%) referred to hospital, and (0.8%) admitted

to the same PHCC. No one of all dirrahael patients died during the study

period.

11. Among (132) diarrheal patients, 12 (9.1%) of enteropathogenic bacteria were

isolated: Salmonella, Campylobacter coli/jejuni, and A. hydrophilia were

isolated in equal numbers from samples 3/12 (25% each), Shigella 2/12

(16.7%), and Y. enterocolytica 1/12 (8.3%). The two Shigella spp. are

Shigella boydii.

12. High number of enteropathogenic bacteria 5/12 (41.7%) were isolated from

Khan Yunis area, 2/12 (16.7%) from northern Gaza, 3/12 (25%) from Gaza

zone, and 2/12 (16.7%) from mid zone.

13. Multidrug resistance was common among Campylobacter coli/jejuni which is

resistant to (52.4%) of antibiotics, followed by A. hydrophilia (49.2%), Y.

enterocolytica (42.9%), Shigella (26.2%), and Salmonella (22.2%).

14. Oral rehydration salts (ORS) is being delivered to (45.8%) of cases, and

(30.3%) of cases obtained antibiotics for diarrhea before culture result were

finished.

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6.2 Recommendations

In light of the results of this study and the above listed conclusions, the following

actions are recommended to minimize antimicrobial resistance and risks of diarrhea:

1. Improvement of laboratories in Gaza strip to increase their ability to isolate

all types of enteropathogenic bacteria especially those, which cannot be

isolated routinely such as Campylobacter spp., A. hydrophilia and Y.

enterocolytica, which require special techniques.

2. Establish a reference laboratory, which is able to identify the serotype of

Salmonella, Shigella and other enteropathogenic bacteria to establish the

epidemiology of these bacteria.

3. Effective strategies to prevent crowding, improve household ventilation,

minimise perennial indoor air pollution, and to improve hygiene.

4. Prevent domestic animals access to the houses especially those capable of

transmitting diseases to peoples including poultry and pigeon.

5. Special attention should be paid to improve the water resource situation in the

regional level.

6. Combat the health problems created by open sewage pools, and cesspool

through the construction of sewage networks especially in Khan Younis to

take all the necessary measures to prevent any environmental risks.

7. Monitor the sanitation facilities, treatment plants, and their impacts on the

underground water.

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8. Conduct studies on the impacts on the public health and raise people's

awareness about the risks and prevention, especially with regard to the health

impacts on children who live near sanitation facilities.

9. Reduce the emergence and spread of antimicrobial-resistant organisms

through reducing the disease burden, spread of infection and education for

physician.

10. Further studies is needed in order to provide a broader picture of the burden

of acute gastroenteritis, antimicrobial-resistant for enteropathogens, risk

factors related to diarrhea in patients in Gaza strip, and study in prescribing

the antibiotics.

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ANNEXE 1

Islamic University-Gaza Master of Biological Sciences Program

Medical Technology

Questionnaire

PHCC Name Code

CASE CONTROL

Diarrhea q yes q no diarrhea within the 3 last

months or birth

Address

Telephone No.

Date of Birth

Age category

_____/_____/__________

q < 5 q > 5

_____/_____/__________

q < 5 q > 5

Gender q male q female q male q female

Refugee status q no q yes q no q yes

No. of household member Total: /________/

No. <5 years old: /__________/

Total: /________/

N. <5 years old: /__________/

No. of rooms in the house /________/ /________/

No. of household workers q none q ≥1

q none q ≥1

Domestic animals in the house

Poultry

Rabbit

Sheep

Pigeon

Donkey

q no q yes

q no q yes

q no q yes

q no q yes

q no q yes

q no q yes

q no q yes

q no q yes

q no q yes

q no q yes

Kitchen equipment

Fridge

Washing machine

Cooking utensils

Latrine in the house

q no q yes

q no q yes

q no q yes

q no q yes

q no q yes

q no q yes

q no q yes

q no q yes

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101

Current water access

Public water access at home

Water from private provider

Roof Jerrican

Filtered water at home

q no q yes

q no q yes

q no q yes

q no q yes

q no q yes

q no q yes

q no q yes

q no q yes

Drinking water

Tap water

Filtered water

Boiled water

Bottle water

Domestic well

Other

q no q yes

q no q yes

q no q yes

q no q yes

q no q yes

q no q yes à /___________/

q no q yes

q no q yes

q no q yes

q no q yes

q no q yes

q no q yes à/____________/

Did your water access change

since January 2009?

q no q yes

q no q yes

Sewage disposal

Barrel

Cesspool

Sewers

q no q yes

q no q yes

q no q yes

q no q yes

q no q yes

q no q yes

Did your sewage disposal

change since January 2009?

q no

q yes

q no

q yes

Clinical data (for case only)

Duration of diarrhea (days) q < 24h q other: /___________/ days

No. of stools the last 24h /___________/ per day

No. of household members reported diarrhea within

10 days before patient’s illness

/_________/

Received antibiotic within 4 weeks before the

beginning of the diarrhea

q no

q yes à name of antibiotic: /______________/

Chronic disease q no q yes à/_________________________/

Fever at attendance time (please check the

temperature)

/_________/ °C

Chills during diarrhea q no q yes

Vomiting during diarrhea q no q yes

Bloody diarrhea q no q yes

Mucous diarrhea q no q yes

Lost of weight q no q yes

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102

Thank you for your cooperation

Researcher

Nahed Abdelateef

Supervisor

Dr. Abdelraouf A. Elmanama

Management (for case only)

Stool culture q no q yes

Antibiotic therapy for diarrhea q no q yes

If yes:

Name of antibiotic: /__________________/

Duration of antibiotic therapy: /__________/ days

Rehydration with ORS q no q yes

Needs to be referred to the hospital q no q yes

If yes à hospital: /________________/

Follow-up (1 week after time admission)

Recovered q no q yes

Persistence of diarrhea q no q yes

Referred to hospital q no q yes

New attendance at the same PHCC q no q yes

Death due to diarrhea q no q yes

Lost to follow-up q no q yes

Stool culture results

Positive

culture

q no q Not performed q yes

q Salmonella

q Shigella

q Yersinia enterocolytica

q Campylobacter

q Aeromonas hydrophila

q Other: /__________________/

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ANNEXE

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104

ANNEXE 3