Detection of Different Diarrheagenic Escherichia coli

Strains by Multiplex PCR.

Dr.Md.Rashedul kabir MBBS

Department of Microbiology Mymensingh Medical College

Mymensingh. Bangladesh 2011

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This thesis entitled �Detection of Different Diarrheagenic Escherichia coli Strains by

Multiplex PCR� is submitted by Dr. Md. Rashedul Kabir in partial fulfillment of the

requirement for the degree of Master of Philosophy (Medical science) in Microbiology,

University of Dhaka. The study was carried out in the Department of Microbiology,

Mymensingh Medical College, Mymensingh during the period of January 2011 to December

2011.

Acceptance of the thesis had been approved by the following board of examiners.

Chairman

Prof. (Dr.) Md. Akram Hossain

Members

1. Prof. (Dr.) Abdullah Akhter Ahmed

2. Prof. (Dr.) Kalpana Deb

3. Prof (Dr.) Marium Khatun

Date of approval, April 30, 2011

Mymensingh.

TO WHOM IT MAY CONCERN

This is to certify that Dr. Md. Rashedul kabir, a student of thesis part has completed the thesis

entitled, �Detection of Different Diarrheagenic Escherichia coli Strains by Multiplex

PCR� in the Department of Microbiology, Mymensingh Medical College, under my

guidance and supervision and this is up to my satisfaction. His protocol was approved by

protocol approval committee of the Department of Microbiology and ethical review

committee of Mymensingh Medical College.

This unbound thesis work is forwarded for kind consideration and needful.

Professor Dr. Md. Akram Hossain Head of the Department of Microbiology Mymensingh Medical College, Mymensingh, Bangladesh.

Declaration

I hereby declare that the whole work submitted as a thesis entitled �Detection of Different

Diarrheagenic Escherichia coli Strains by Multiplex PCR� in the Department of the

microbiology, Mymensingh Medical College, Dhaka University, for the Degree of Master of

philosophy is the result of my own investigation and was carried out under the supervision of

Professor Dr. Md. Akram Hossain.

I, further declare this thesis or part thereof has not been concurrently submitted for the award

of any Degree or Diploma anywhere.

Dr. Md. Rashedul kabir M Phil, Thesis part Department of Microbiology Mymensingh Medical College Mymensingh.

ACKNOWLEDGEMENT

Thanks to Almighty Allah, the merciful and the beneficial for giving me the opportunity

courage and enough energy to carry out and complete the entire thesis work.

I am grateful and deeply indebted to my guide Professor Dr. Md. Akram Hossain, Head of

the Department of Microbiology, Mymensingh Medical College, Mymensingh. I have the

great privilege and honour to express my indebtedness to him for kindly placing at my

disposal all the facilities available in the department, for his guidance, supervision, inspiring

encouragement, constructive criticism and help in carrying out this thesis work.

I am grateful and express thanks to the honorable members of the Ethical Review Committee

for giving kind approval to my research protocol. I am obliged to Professor Dr. Md. Aminul

Hoque, Principal of Mymensingh Medical College, Mymensingh for his kind permission to

conduct the thesis.

My cordial respect and complements to Dr. Shyamal Kumar Paul, Assistant Professor, of

Microbiology, Mymensingh Medical College, Mymensingh for his help from the beginning

of the course up to completion of my thesis.

I owe my gratitude to respective teacher Dr. Md Chand Mahamud, Assistant Professor of

Microbiology, Mymensingh Medical College, Mymensingh, for his constructive criticism in

correcting this thesis with his wise advice and active cooperation.

I am grateful and express thanks to Professor Nobumichi Kobayashi, Sapporo Medical

University of Japan for providing with me primers for PCR and for his valuable suggestion.

I specially extend my heartfelt gratitude to Dr. Azizul Haque, Associate Professor, of

Pediatrics�, Mymensingh Medical College, Mymensingh for his cordial cooperation and

valuable suggestion.

I like to express gratitude to all other teachers, laboratory technologist and other staffs of

Microbiology department, Mymensingh Medical College, Mymensingh for their constant

help and sincere cooperation during the entire study period.

I express my gratitude to my parents, friends and well wisher who inspire and help me

continuously.

I remained incomplete if I do not express whole hearted thanks and gratitude to my wife

Dr. Tasnuva Mariom for sparing me so much time in this job from all sorts of social and

family responsibilities and sharing my pain and pleasure. Last but not least I express my

heartiest affection for my kid Tahsan who had been deprived of my care and attention during

the thesis work.

Lastly I am indebted to all those persons from whom I have collected samples. May Allah

give them better rewards. Thank again.

Dr. Md. Rashedul Kabir

Dedicated

to

Professor Dr. Md. Akram Hossain

and

My parents

CONTENTS

Page No.

LIST OF TABLES VIII

LIST OF FIGURES IX

LIST OF ABBREVIATIONS X

SUMMARY XI-XII

CHAPTER 1 : INTRODUCTION & OBJECTIVES 1 � 5

INTRODUCTION 1

OBJECTIVES 5

CHAPTER 2 : REVIEW OF LITERATURE 6 � 40

CHAPTER 3 : MATERIALS AND METHODS 41 � 51

CHAPTER 4 : RESULTS 52 � 65

CHAPTER 5 : DISCUSSION 66 � 74

CHAPTER 6 : CONCLUSION & RECOMMENDATIONS 75

LIST OF REFERENCES 76 � 104

PHOTOGRAPHS 105 � 115

APPENDICES i� xiii

LIST OF TABLES

Table No. Title Page No.

I Age and Sex distribution of study population. 53

II Distribution of enteropathogens in the stool samples. 55

III Distribution of different diarrheagenic Escherichia

coli (DEC) Strains in the study population.

57

IV Distribution of different genes (ST / LT/ Both) of

Enterotoxogenic Escherichia coli (ETEC) strains in

the study population.

58

V Detection rate of Enterotoxogenic Escherichia coli

(ETEC) in different age group of the study population.

59

VI Detection rate of Enteroaggregative Escherichia coli

(EAEC) in different age group of the study population

60

VII Clinical features of Enterotoxogenic Escherichia coli

(ETEC) in the study population.

62

VIII Clinical features of Enteroaggregative Escherichia coli

(EAEC) in study population.

63

IX Antibiotic sensitivity patterns of Enterotoxogenic

Escherichia coli (ETEC) isolated from the stool

samples.

64

X Antibiotic sensitivity patterns of Enteroaggregative

Escherichia coli (EAEC) isolated from the stool

samples.

65

LIST OF FIGURES

Figure No.

Title

Page No.

1.1 Age distribution of study population 54

1.2 Sex distribution of study population 54

LIST OF ABBREVIATIONS

A/A Aggregative adherence

A/E Attaching and effacing

AID Acute Infectious Diarrhea

ATCC American Type Culture collection.

CDC Centre for Disease Control and Prevention

CFU Colony forming unit

CLSI Clinical and Laboratory Standards Institute

DNA Deoxyribonucleic acid

dNTP Deoxynucleoside triphosphate

EIA Enzyme immune assay

ELISA Enzyme Linked Immunosorbent Assay

Ial Invasion-associated locus

LEE Locus of enterocytes effacement

LPS Lipopolysaccharide

LT Heatlabile toxin

PAGE Polyacrylamide Gel electrophoresis

PCR Polymerase Chain Reaction

RNA Ribonucleic acid

SMAC Sorbitol Mac Con key

ST Heat-stable toxin

Taq Thermus aquaticus

TBE Trise bouric acid EDTA buffer

WHO World Health Organization

SUMMARY

Background: Diarrhaegenic Escherichia coli is one of the most important etiologic agent of

acute diarrhea and represent a major public health problem in developing countries like

Bangladesh. Due to lack of facilities diarrhaegenic Escherichia coli can not be detected in the

routine diagnostic microbiology laboratory. Recently various multiplex PCR methods have

been developed to detect diarrheagenic Escherichia coli. Thereby, the present study was

conducted in the Department of Microbiology, Mymensingh Medical College to detect

different diarrheagenic Escherichia coli strains by multiplex PCR.

Methods: This cross sectional study was done during the period from January 2011 to

December 2011 and included all patients with acute diarrhea irrespective of age and sex

admitted in Mymensingh Medical College Hospital and S.K hospital, Mymensingh. A total of

300 stool samples were examined by standard laboratory methods for identification of

enteropathogens. Different diarrheagenic Escherichia coli strains were detected by Multiplex

PCR following standard methods. Rotavirus was detected by Polyacrylamide Gel

electrophoresis (PAGE).

Results: Of the 300 stool samples examined, Enteropathpgens were detected in 160 (53.5%)

cases. Rotavirus was detected in 82 (27.5%) cases, followed by diarrheagenic Escherichia

coli (DEC) in 54 (18%), Shigella spp. in 8 (2.4%), Salmonella spp. in 5 (1.6%), E. Histolytica

in 4 (1.5%) and G. Lamblia in 3 (1.0%) cases. Among the diarrheagenic Escherichia coli

(DEC), Enterotoxogenic Esch.coli (ETEC) was detected in 39 (13%) cases and

Enteroaggregative Esch .coli (EAEC) in 15 (5%) cases. No Enterohemorrhagic Esch .coli

(EHEC), Enteropathogenic Esch .coli (EPEC) and Enteroinvasive Esch .coli (EIEC) could be

detected. Both (ST and LT) genes of ETEC were detected in 26 (67%) cases, ST gene in 9

(23%) cases and LT gene in 4 (10%) cases.

Conclusion: Analyzing the findings of this present study, diarrheagenic Escherichia coli was

found to be one of the most important cause of acute diarrhea. Detection rate of diarrheagenic

Escherichia coli by multiplex PCR method was quite satisfactory. Thereby, Multiplex PCR

could be adapted to detect different diarrheagenic Escherichia coli in setup like Medical

colleges or tertiary medical facilities.

Introduction

Acute infectious diarrhea (AID) is a major cause of morbidity and mortality worldwide, and

it remains a major public health challenge especially in developing countries where it is a

leading cause of death. Every year nearly 1.4 billion episodes of AID occur in children less

than 5 years of age in developing countries (Kosek et al. 2003; Parashar et al. 2003), of which

123.6 million episodes required outpatient medical care and 9 million episodes required

hospitalization. It has been estimated that the mean number of episodes of diarrhea per year

in children under 5 years of age from a developing region is 3.2, with the highest incidence

(4.8 episodes), occurring during the first year of life, decreasing progressively to 1.4 episodes

per year at 4 years of age. Furthermore, the highest age-mortality rate (8.5 children per

1000/year) occurred in children under 1 year of life (Kosek et al. 2003). In addition diarrheal

illness account for an estimated 12600 deaths each day in children under 5 years of age in

Asia, Africa and Latin America (Nguyen et al. 2005).

Kosek and associates (2003) calculated studies from 1990 to 2000 and concluded that

diarrhea accounts for 21% of all deaths at under five years of age causing 2.5 million death

per year in developing countries. Improvements in sanitation, nutrition, education and early

access to oral rehydration therapy among other measures have lowered the lethality of severe

AID from 4.6 million in 1982 to an estimated 1.5�2.5 million in 2010 (Black et al. 2010;

O'Ryan et al. 2010). However, it remains the second most common cause of death in children

under 5 years of age worldwide according to the Global Burden Disease Report of the WHO

(Kosek et al. 2003).

A diversity of recognized microorganisms such as bacteria, viruses and parasites can be

associated with severe AID in children (Al-Gallas et al. 2007; O'Ryan et al. 2005). However,

etiological information that includes all the known agents of severe AID in children is scarce.

Numerous studies performed in different countries have reported diarrheagenic Escherichia

coli (DEC) pathotypes as being the most frequent and important among bacterial pathogens

associated with AID in developing countries. However, the frequencies of these pathogens

vary with geographic region and depend on the socioeconomic/sanitary conditions achieved

(Black et al. 2010; O'Ryan et al. 2010). Altogether, they represent 30-40% of the cases

(Albert et al. 1999), and about 15-30% have required hospital care (Nataro & Karper 1998;

O'Ryan et al. 2005).

In Bangladesh, the AID remains one of the most important health problems surpassed only by

the respiratory diseases. One third of the total child death burden is due to diarrhea. Every

year, a rural child suffers on average from 4.6 episodes of diarrhea, from which about

230,000 children die (Piechulek et al. 2003). Diarrheagenic Escherichia coli has been

reported to be responsible for 34% of diarrheal episodes in Bangladesh (ICDDR, B 2002).

Escherichia coli is the predominant nonpathogenic facultative anaerobic member of the

human intestinal micro flora. Some Escherichia coli strains, however have developed the

ability to cause disease of the gastrointestinal, urinary and central nervous systems in the

human host. Escherichia coli has been implicated as an agent of diarrheal disease since the

1920s (Nguyen et al. 2005).

There are now at least six types of diarrheagenic strains of Escherichia coli on the basis of

distinct epidemiology and clinical feature, special virulence determinants and association

with certain serotypes: Enterotoxogenic Escherichia coli (ETEC), Enteropathogenic

Escherichia coli (EPEC), Enteroheamorragic Escherichia coli (EHEC), Enteroaggregative

Escherichia coli (EAEC), Enteroinvasive Escherichia coli (EIEC) and Diffusely adhering

Escherichia coli (DAEC). Of these EPEC, ETEC, EIEC, EHEC and EAEC are clearly

associated with different types of enteritis, while the another pathotype is candidate as

potential pathogens but their association with diarrhea has not been clearly assessed and

further studies are required to confirm their etiological role in diarrheal diseases (Rappelli et

al. 2005).

Due to lack of facilities diarrhaegenic Escherichia coli can not be detected in the routine

diagnostic microbiology laboratory in developing countries, which is important in

understanding the disease spectrum, tracing the sources of infection and the burden of the

disease. Such identification would also assist the clinician to dispense appropriate

management (Nessa et al. 2007).

Identification of diarrheagenic Escherichia coli can not be done only by culture and

biochemical tests, since they are indistinguishable from the non pathogenic Escherichia coli

commonly found in human feces. Moreover, specific serotyping is not always correlated with

pathogenicity. The discrimination of different diarrheagenic Escherichia coli strains requires

DNA target based screening by molecular techniques (Rappelli et al. 2005).

PCR, one of the molecular based detection methods, commonly used for rapid and reliable

diagnosis, which has a high sensitivity and specificity. To detect different diarrheagenic

Escherichia coli strains, it is necessary to run several individual PCRs with different primer

pairs. In order to simplify diagnosis, various multiplex PCR methods have been developed

for the simultaneous detection of several specific genes of different diarrheagenic

Escherichia coli strains. This method showed high sensitivity and high specificity for

identification of human diarrheagenic Escherichia coli (Toma et al. 2003).

The objective of the present study is to evaluate the multiplex PCR as a diagnostic tool for

different diarrheagenic Escherichia coli.

OBJECTIVES

General objective:

To evaluate the molecular technique for detecting different diarrheagenic Escherichia coli

strains by Multiplex PCR.

Specific objectives:

1. To isolate and identify Escherichia coli by culture and different biochemical tests.

2. To detect different diarrheagenic Escherichia coli strains by amplifying specific genes by

Multiplex PCR.

3. To determine the prevalence of different diarrheagenic Escherichia coli Strains and other

enteropathogens in the study population.

4. To find out antimicrobial sensitivity patterns of isolated strains.

REVIEW OF LITERATURE

Acute Infectious Diarrhea (AID) � A worldwide problem

Acute diarrhea is a clinical condition defined as an increased frequency of defecation (three

or more times per day or at least 200 g of stool per day ) lasting less than 14 days, may be

accompanied by nausea, vomiting, abdominal cramping, clinically significant systemic

symptoms, or malnutrition (Thieman et al. 2004; WHO, 2005). Acute infectious diarrhea

(AID) is a major cause of morbidity and mortality worldwide, and it remains a major public

health challenge especially in developing countries where it is a leading cause of death. Every

year nearly 1.4 billion episodes of AID occur in children less than 5 years of age in

developing countries, of which 123.6 million episodes require outpatient medical care and 9

million episodes require hospitalization (Kosek et al. 2003; Parashar et al. 2003). It has been

estimated that the mean number of episodes of diarrhea per year in children under 5 years of

age from a developing region is 3.2, with the highest incidence (4.8 episodes), occurring

during the first year of life, decreasing progressively to 1.4 episodes per year at 4 years of

age. Furthermore, the highest age-mortality rate (8.5 children per 1000/year) occurred in

children under 1 year of life (Kosek et al. 2003). In addition diarrheal illness account for an

estimated 12600 deaths each day in children under 5 years of age in Asia, Africa and Latin

America (Nguyen et al. 2005).

Kosek and associates (2003) calculated studies from 1990 to 2000 and concluded that

diarrhea accounts for 21% of all deaths at under five years of age causing 2.5 million deaths

per year in developing countries. Improvements in sanitation, nutrition, education and early

access to oral rehydration therapy among other measures have lowered the lethality of severe

AID from 4.6 million in 1982 to an estimated 1.5�2.5 million in 2010 (Black et al. 2010).

However, it remains the second most common cause of death in children under 5 years of age

worldwide according to the Global Burden Disease Report of the WHO (Kosek et al. 2003).

A diversity of recognized microorganisms such as bacteria, viruses and parasites can be

associated with severe AID in children (Al-Gallas et al. 2007; O'Ryan et al. 2005). Numerous

studies performed in different countries have reported diarrheagenic Escherichia coli (DEC)

as being the most frequent and important among bacterial pathogens associated with AID in

developing countries. However, the frequencies of these DEC vary with geographic region

and depend on the socioeconomic/sanitary conditions achieved (Black et al. 2010).

Altogether, they represent 30-40% of the cases (Albert et al. 1999), and about 15-30% have

required hospital care (Nataro & Karper 1998; O'Ryan et al. 2005).

In Bangladesh, the AID remains one of the most important health problems surpassed only by

the respiratory diseases. One third of the total child death burden is due to diarrhea. Every

year, a rural child suffers on average from 4.6 episodes of diarrhea, from which about

230,000 children die (Piechulek et al. 2003). Diarrheagenic Escherichia coli has been

reported to be responsible for 34% of diarrheal episodes in Bangladesh (ICDDR, B 2002).

ESCHERICHIA COLI

Escherichia coli (E. coli) is a Gram-negative, rod-shaped bacterium that is commonly found

in the lower intestine of warm-blooded animals. Most E. coli strains are harmless, but some

serotypes can cause serious food poisoning in humans (CDC 2011). The harmless strains are

part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2, and

by preventing the establishment of pathogenic bacteria within the intestine (Reid et al. 2001).

E. coli and related bacteria constitute about 1% of gut flora (Eckburg et al. 2005), and fecal-

oral transmission is the major route through which pathogenic strains of the bacterium cause

disease. Cells are able to survive outside the body for a limited amount of time, which makes

them ideal indicator organisms to test environmental samples for fecal contamination (Feng

et al. 2002). The bacterium can also be grown easily and inexpensively in a laboratory

setting, and has been intensively investigated for over 60 years. E. coli is the most widely

studied prokaryotic model organism, and an important species in the fields of biotechnology

and microbiology, where it has served as the host organism for the majority of work with

recombinant DNA (Nataro & Karper 1998).

History The scientific history of Escherichia coli (E. coli) started with its first description in 1885 by

Theodor von Escherichia (Escherichia 1988; Gross & Rowe 1985), a pediatrician and

scientist, who in a series of pioneering studies of the intestinal flora of infants discovered a

normal microbial inhabitant of healthy individuals that he named Bacterium coli commune.

Its actual name became used and officially accepted in 1958, in his honor (Kuhnert et al.

2000).

Taxonomy

Domain: Bacteria, Kingdom: Bacteria, Phylum: Proteobacteria, Class: Gamma

Proteobacteria, Order: Enterobacteriales, Family: Enterobacteriaceae, Genus: Escherichia,

Species: Escherichia coli (Wikipedia 2008).

Structure of E.coli

The E. coli cell is about 1 ìm in diameter. It has a protective outer membrane and an inner

plasma membrane that encloses the cytoplasm and the nucleoid. Between the inner and outer

membranes is a thin but strong layer of peptidoglycans (sugar polymers cross-linked by

amino acids), which gives the cell its shape and rigidity. The plasma membrane and the

layers outside it constitute the cell envelope. Differences in the cell envelope account for the

different affinities for the dye Gentian violet, which is the basis for Gram's stain; gram-

positive bacteria retain the dye, and gram-negative bacteria do not (Nataro & Karper 1998;

Chakraborty, P 2003; Wikipedia 2011).

From the outer membrane of E. coli cells and some other eubacteria protrude short, hair- like

structures called pili, by which cells adhere to the surfaces of other cells. Strains of E. coli

have long peritrichous flagella, which can propel the bacterium through its aqueous

surroundings. Bacterial flagella are thin, rigid, helical rods, 10 to 20 nm thick. They are

attached to a protein structure that spins in the plane of the cell surface, rotating the flagellum

(Chakraborty, P 2003; Wikipedia 2011).

The cytoplasm of E. coli contains about 15,000 ribosomes, thousands of copies of each of

several thousand different enzymes, numerous metabolites and cofactors, and a variety of

inorganic ions. Under some conditions, granules of polysaccharides or droplets of lipid

accumulate. The nucleoid contains a single, circular molecule of DNA. Although the DNA

molecule of an E. coli cell is almost 1,000 times longer than the cell itself, it is packaged with

proteins and tightly folded into the nucleoid, which is less than 1 ìm in its longest dimension.

As in all bacteria, no membrane surrounds the genetic material. In addition to the DNA in the

nucleoid, the cytoplasm of most bacteria contains many smaller, circular segments of DNA

called plasmids. These nonessential segments of DNA are especially amenable to

experimental manipulation and are extremely useful to the molecular geneticist. In nature,

some plasmids confer resistance to toxins and antibiotics in the environment (Wikipedia

2011; Nataro & Karper 1998).

Biology and biochemistry

The species Escherichia coli comprises Gram-negative, rod-shaped, non-spore forming,

motile bacteria which are about 1 ìm in diameter, with a cell volume of 0.6-0.7 ìm3

(Darnton et al. 2007; Kubitschek 1990). They are facultative anaerobes, oxidase-negative,

glucose, lactose and sucrose fermenting, with an optimum growth pH of 6.0-7.0 and

temperature of 37°C. However, some laboratory strains can multiply at temperatures up to

49°C (Fotadar et al. 2005).

E. coli and related bacteria possess the ability to transfer DNA via bacterial conjugation,

transduction or transformation, which allows genetic material to spread horizontally through

an existing population. This process led to the spread of the gene encoding shiga toxin from

Shigella to E. coli O157:H7, carried by a bacteriophage (Brüssow et al. 2004).

Serotypes

In 1944, Kauffman proposed a scheme for the serologic classification of E. coli which is still

used in modified form today. According to the modified Kauffman scheme, E. coli are

serotyped on the basis of their O (somatic), H (flagellar), and K (capsular) surface antigen

profiles. Although more than 175 O antigens and 53 H antigens are currently recognized, the

number of serotype combinations responsible for diarrheal disease is small (Nataro & Karper

1998).

Genomes

The first complete DNA sequence of an E. coli genome was published in 1997. It was found

to be a circular DNA molecule 4.6 million base pairs in length, containing 4288 annotated

protein-coding genes, seven ribosomal RNA (rRNA) operons, and 86 transfer RNA (tRNA)

genes. Despite having been the subject of intensive genetic analysis for approximately 40

years, a large number of these genes were previously unknown. The coding density was

found to be very high, with a mean distance between genes of only 118 base pairs. The

genome was observed to contain a significant number of transposable genetic elements,

repeat elements, cryptic prophages, and bacteriophage remnants (Blattner et al. 1997).

Today, over 60 complete genomic sequences of Escherichia species are available.

Comparison of these sequences shows a remarkable amount of diversity; only about 20% of

each genome represents sequences that are present in every one of the isolates, while

approximately 80% of each genome can vary among isolates. The total number of different

genes among all of the sequenced E. coli strains (the pan-genome) exceeds 16,000. This very

large variety of component genes has been interpreted to mean that two-thirds of the E. coli

pan-genome originated in other species and arrived through the process of horizontal gene

transfer (Zhaxybayeva et al. 2011).

Virulence Factors

Several highly adapted strains have acquired virulence factors that allow them to cause

disease. In addition to structural properties that allow survival in the gastrointestinal tract

(Foster 2004), virulence factors include:

i. Adhesin / colonization factors (specific adherence factors that allow colonization of

atypical sites for E coli):

A) Fimbriae (pili) or fibrillae aid cell surface binding.

B) Outer membrane surface structures can trigger signal transduction pathways or

cytoskeletal rearrangements that can lead to illness.

C) Proteins that bind to cell surface receptors can trigger cytokine cascades (eg,

lipopolysaccharide, flagellin)

ii. Toxins (secreted toxins and other effectors proteins):

Heat-labile enterotoxin (LT) activates adenylate cyclase which eventually leads to chloride

secretion and diarrhea. LT-producing strains induce watery diarrhea among adults in Asia,

travelers to Central America, and children in several regions (Guerrant 1980; Guerrant 2005).

Heat-stable enterotoxin a (STa) activates guanylate cyclase that provokes ion secretion,

which leads to diarrhea. Heat-stable enterotoxin b (STb) activates intracellular calcium that

stimulates ion secretion, which leads to diarrhea (Guerrant 1980; Guerrant 2005).

Cytotoxins (Shiga-like toxin SLT-1, SLT-2) cause hemorrhagic colitis or hemolytic uremic

syndrome (HUS). SLT-1 is virtually identical to Shiga toxin, which is produced by Shigella

dysenteriae type 1 and SLT-2 also is very similar to the Shiga toxin molecule. SLT 1 or 2 are

encoded on a lambda-like bacteriophage; the ability to produce SLT was a key step in the

evolution of EHEC from EPEC (Reid 2000; Strockbine 1986).

iii. Type 2 secretion systems (T2SSs) :

T2SSs are comprised of about 12 proteins that form a piston-like structure for exporting

proteins and toxins across the outer membrane. These systems are encoded on pathogenicity

islands (segments of chromosomal DNA that are flanked by insertion or repeat elements)

or on plasmids (Clarke 2003 ; Lathem 2002).

iv. Type 3 secretion systems (T3SSs):

T3SSs contain genes encoding proteins that form a "molecular syringe" for injection of

bacterial proteins or toxins. As with T2SSs, these systems can be encoded on pathogenicity

islands or on plasmids (Donneberg 2005).

v. Plasmids:

Plasmids are genetic elements that can be transmitted between bacteria. They are not

virulence factors, but they can encode genes for a variety of factors that contribute to

pathogenesis, including antibiotic resistance, fimbriae, toxins, secretion systems, and invasion

factors. Transmission of plasmids plays a large role in the growing problem of antibiotic

resistance (Prats 2003).

Diarrheagenic Escherichia coli

The pathogenic E. coli species comprise a very versatile group with numerous virulence

determinants (virulence factors) including adhesins, invasins, toxins and secretion systems

that allow them to act as causative agents in both human and veterinary medicine (Kaper et

al. 2004; Nataro & Karper 1998). In human, these pathogens are responsible for three main

types of clinical infections: (i) enteric or diarrheal diseases, (ii) urinary tract infections, and

(iii) meningitis/septicemia. Based on their distinct virulence properties and clinical symptoms

of the host, pathogenic E. coli strains are divided into numerous categories or pathotypes:

Diarrheagenic Escherichia coli (DEC); Uropathogenic Escherichia coli (UPEC); Neonatal

meningitis/sepsis associated Escherichia coli (NMEC) (Dawson et al. 1999; Kaper et al.

2004).

Diarrheagenic Escherichia coli (DEC) belong to one of the most frequent, interesting and

widespread versatile pathogenic groups of bacteria that cause severe disease in human

(Campos et al. 2004; Nataro & Karper 1998). DEC has been divided into six well-

characterized subgroups or classes [enterotoxigenic E. coli (ETEC), enteropathogenic E. coli

(EPEC), verocytotoxin producing E. coli (VTEC) or enterohemorrhagic E. coli (EHEC),

enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), diffusely adherent E. coli

(DAEC)] based on clinical manifestations, phenotypic traits, specific virulence properties,

and pathogenesis (Kaper et al. 2004; Nataro & Karper 1998).

Enterotoxigenic E. coli (ETEC)

ETEC is one of the most studied pathotypes of DEC. Most studies have demonstrated the

association of ETEC with diarrhea among infants less than five years of age (Aguero et al.

1985; Shaheen et al. 2004). Most of the illness, both in terms of number of cases and severity

of symptoms, occur in infants after weaning. Moreover, epidemiological studies have

implicated contaminated food and water as the most common vehicles of ETEC infection.

Thus, sampling of both food and water sources from endemic areas have demonstrated the

spread of ETEC bacteria in the community (Black et al. 1982). A high infectious dose

(ranging 106 to 1010 CFU) of ETEC has been demonstrated in human (Levine et al.1979;

Nataro & Karper 1998), and a large number of serogroups of ETEC have been associated

with diarrhea (Stenutz et al. 2006).

ETEC strains cause cholera-like watery diarrhea through the elaboration and action of LT

(heat-labile) and/or ST (heat-stable) enterotoxins. The ability of ETEC strains to produce

diarrheal illness by either or both of theses enterotoxins is what defines an ETEC (Nataro &

Karper 1998).

However, to cause diarrhea, by LT and/or ST, the ETEC must adhere and colonize the

intestinal mucosa. This is achieved by attaching with one or more colonization factor antigens

(CFAs), which are antigenically diverse and usually are encoded by plasmids (Nataro &

Karper 1998; Smith et al.1983). LTs of ETEC are oligomeric toxins that are functionally and

structurally related to the cholera toxin (CT) expressed by Vibrio cholerae, serogroups O1

and O139 (Sixma et al. 1993).

There are two types of LTs, LT-I and LT-II that are commonly found in human and animal

isolates. However, the term LT refers to LT-I, which is associated with disease in both human

and animals, while LT-II is expressed only in animals, but it is rarely associated to disease

(Nataro & Karper, 1998; Smith et al. 1983). LT-I is constituted by ~80% amino acid identity

with CT that consists of a single A subunit and five identical B subunits. The A subunit is

responsible for the enzymatic activity, and the B subunits are responsible for the toxin

binding to the cell surface ganglioside. After endocytosis the A subunit stimulates a series of

intracellular processes leading to increased level of cyclic adenosine monophosphate

(cAMP), resulting in an increased phosphorylation of chloride channels, and hence a reduced

absorption of NaCl. This increased extracellular ions content results in osmotic diarrhea.

Besides, there is an increased secretion of ions in the villi crypts (Nataro & Karper 1998).

STs are small peptides including two unrelated classes, STa and STb, which differ in

structure and mechanism of action. Genes for both classes � estA and estB � have been found

either on plasmids or on transposons. Only toxin of the STa class has been associated with

human and animal diseases. It has been established that the STa receptor is located on the

apical surface of enterocytes and that binding to the receptors leads to increased intracellular

cyclic guanidine monophosphate (cGMP) levels, which affects the electrolytic balance in a

similar manner as LT (Kaper et al. 2004; Nataro & Karper 1998). In contrast, STb is

primarily associated with diarrhea in piglets; however, some human ETEC isolates

expressing STb have been reported (Nataro & Karper 1998; Schulz et al. 1990). STb can

elevate cytosolic Ca+2 concentrations, stimulating both the release of prostaglandin E2 and

serotonin, which lead to increased ion secretion (Kaper et al. 2004; Nataro & Karper 1998).

The clinical features of ETEC diarrhea are constant with the pathogenic mechanisms of its

enterotoxins, and characterized by watery diarrhea without blood, mucus or pus, fever and

vomiting (DuPont 2009). The illness is typically abrupt, but can vary from mild, brief, and

self-limiting to a severe disease similar to in Vibrio cholerae infection (Levine et al. 1977).

Several studies have showed that the percentage of ETEC infection varies from 10 to 30%

and the clinical presentation, as with the other DEC varies among different geographical

areas (Mangia et al. 1993).

Enteropathogenic E. coli (EPEC)

EPEC was the first pathotype of DEC described, and associated to the infant diarrheal

diseases worldwide (Nataro & Karper 1998). The characteristic intestinal histopathology �

attaching and effacing (A/E) lesions - of EPEC are associated to striking cytoskeletal changes

in the epithelial cell. This ability to induce-attaching and effacing lesions is encoded by genes

for the adherence factor intimin (eae), a type II secretion system (TTSS) that includes the esc

genes and the translocated intimin receptor (Tir) (Nataro & Karper 1998) .These genes are

located on a 35-Kb (kilobase) pathogenicity island (PAI), called the locus of enterocyte

effacement (LEE), which is present in all EPEC and EHEC (McDaniel et al. 1995).

The A/E mechanism induce microvilli destruction, intimate adherence of bacteria to the

intestinal epithelium, pedestal formation, and aggregation of polarized actin and other

elements of the cytoskeleton at the site of bacterial attachment (McDaniel et al. 1995; Nataro

& Karper 1998). In addition, an EPEC virulence factor outside the LEE has also been

described (Trabulsi et al. 2002), which is located on a plasmid encoding a type pilus

called bundle forming pilus (bfp). Besides, on another plasmid there is a plasmid encoded

regulator gene (per) which regulates several chromosomal and plasmid genes necessary for

the pathogenesis of A/E lesions (Levine et al. 1985)).Beside the virulence factors described

above, there are other potential virulence factors such as EAST1 (enteroaggregative E. coli

heat-stable enterotoxin1), originally indentified in Enteroaggregative E. coli strains, but its

significance in pathogenesis is uncertain (Nataro &Karper 1998).

Based on these properties, two variants of EPEC � typical and atypical EPEC � have been

reported (Hien et al. 2007) and their prevalence seems to be different between developing and

developed countries. The typical EPEC is a cause of diarrhea in developing countries, while

atypical EPEC seems to be associated with diarrhea in developed countries (Afset et al.

2003).

The most special feature of the epidemiology of disease due to EPEC infection is the

remarkable age distribution. EPEC infection is primarily a disease of infants younger than 2

years of age. EPEC infection, as with other DEC pathotypes, takes place via the faecal-oral

route with contaminated food and water. The infectious dose among infants is not known, but

is presumed to be much lower than 108 CFU/microorganism (Nataro & Karper 1998).

The clinical features of EPEC infection are characterized by watery diarrhea, vomiting and

low-grade of fever. As compared to the developed countries, EPEC infection plays a more

important role in developing countries, where it has been found as one of the foremost causes

of diarrhea (Cravioto et al. 1988). EPEC may also cause diarrhea under other circumstances,

such as nosocomial outbreaks.

Enteroaggregative E. coli (EAEC)

EAEC was first described in 1985, recognized by its distinctive adherence to HEp-2 cells in

an aggregative, �stacked brick-like� pattern (Nataro et al. 1998; Pereira et al. 2008). This

adherence pattern, distinguishable from the adherence patterns manifested by EPEC and

DAEC, was first significantly associated with diarrhea among Chilean children in 1987

(Nataro et al. 1987). EAEC is defined as an E. coli pathotype that does not secrete heatlabile

or heat-stable enterotoxins but adheres to HEp-2 cells and other epithelial cells in an

aggregative adherence (A/A) pattern, the genes for which are encoded on plasmids (Nataro et

al. 1998).

Many epidemiological studies have used the A/A pattern and the plasmid-encoded probe

�CVD432� or simply the A/A probe to identify EAEC (Baudry et al. 1990). Moreover, a

transcription activator known as �AggR�, the gene which regulates the A/A genes has been

described as the major EAEC virulence regulator (Nataro 2005). Recently, some

epidemiological studies have suggested that CVD432-positive strains, which are predicted to

carry the AggR regulators, are the true EAEC pathogens termed �typical EAEC� (Harrington

et al. 2006). However, A/A probe negative isolates share virulence factors with A/A probe

positive isolates, a finding which indicates that additional factors are involved in the A/A

phenotype in these EAEC strains (Bouzari et al. 2001) .

The pathogenesis of EAEC is complex as the strains are relatively heterogeneous. However

three major features of the EAEC infections have been suggested as basic strategy of EAEC:

(i) abundant adherence to the intestinal mucosa (colonization), (ii) elaboration and secretion

of enterotoxins and cytotoxins, and (iii) induction of mucosal inflammation (Harrington et al.

2006; Nataro et al. 1998). Furthermore, several studies have reported the presence of fecal

lactoferrin and proinflammatory cytokines, notably interleukin (IL)-8 (Greenberg et al. 2002),

as well as the presence of a putative virulence factors, such as yersinia-bactin lectin (Basu et

al. 2004), in patients infected with EAEC.

Since its discovery, an increasing number of studies have associated EAEC with diarrhea in a

variety of settings. These include endemic diarrhea of infants in both industrialized and

developing countries (Elias et al. 1999), persistent diarrhea among acquired

immunodeficiency syndrome patients and traveler�s diarrhea (Jenkins et al. 2006). The

implication of EAEC in diarrheal outbreaks confirmed the fact that at least some strains

exhibiting the A/A phenotype were true human pathogens, yet not in all studies were EAEC

associated with diarrheal illness (Nataro et al. 1995). Pathogenesis studies of EAEC

experienced a step-forward in 1994, when the first EAEC volunteer study was published

(Nataro et al. 1995). In this report, it was showed that only one of four A/A probe positive

EAEC strains elicited human diarrhea, confirming that not all EAEC strains were equally

pathogenic.

Although not all EAEC strains have been associated to diarrheal disease, the most commonly

reported symptoms associated with EAEC infection are watery diarrhea with or without

blood and mucus, abdominal pain, nausea, vomiting, and low-grade fever (Adachi et al.

2002). Besides, EAEC can cause both an acute and a chronic (>14 days) diarrheal illness in

different parts of the world. A growing number of studies from both developing and

developed countries have supported the association of EAEC with persistent diarrhea in

young children and adults (Araujo et al. 2007). It has also been reported that EAEC may

be a leading cause of outbreaks of acute diarrheal disease affecting newborns and children

(Cohen et al. 2005). EAEC have been reported as the second most common bacterial

pathogen isolated in US adult travelers to developing countries and deployed US military

personnel (Adachi et al. 2002).

In another study on adult US travelers to Mexico that evaluated the serologic response to the

EAEC anti-aggregative protein dispersin, 48% of the travelers developed increases in

antibody levels over time; the majority of patients, though, remained asymptomatic (Huang et

al. 2008). These findings may possibly suggest that EAEC infection shows a variety of

clinical presentations and support the idea of a great heterogeneity of EAEC as a virulent

pathogen (Nataro et al. 1995).

Enterohemorrhagic E. coli (EHEC)

EHEC is an etiological agent of diarrhea with life-threatening complications. EHEC belongs

to a group of E. coli called VTEC (�verotoxigenic E. coli� or �Verocytotoxin-producing E.

coli�) or STEC (�Shiga toxin-producing E. coli), formerly SLTEC (�Shiga-like

toxinproducing E. coli�). EHEC colonize the intestinal mucosa inducing a characteristic

attaching and effacing (A/E) lesion, which is also present in EPEC and EAEC. The classic

intestinal histopathology characteristic of EHEC infection includes hemorrhage and edema in

the lamina propria, which results in bloody diarrhea, hemorrhagic colitis, necrosis and

intestinal perforation. The major virulence factor, and a defining characteristic of EHEC, is

the Shiga toxin (Stx), a potent cytotoxin that leads to cell death and will aggravate the

symptoms in patients infected (Nataro & Karper 1998).

Most studies have reported EHEC strains as important pathogens causing diarrhea, with

association to Hemorrhagic Colitis (HC) and Hemorrhagic Uremic Syndrome (HUS), which

are due to the interaction of Shiga toxin (Stx) with endothelial cells (Brown et al.1989). The

term �enterohemorrhagic E. coli� (EHEC) was originally created to denote strains that cause

HC and HUS, express Stx, cause A/E lesions on epithelial cells, and possess a plasmid

(Levine 1987). Thus, EHEC denotes a subset of STEC and includes a clinical connotation

that is not implied with STEC. Whereas not all STEC strains are believed to be pathogens, all

EHEC strains by the above definition are considered pathogens (Levine 1987).

The Stx family contains two major, immunologically non-cross-reactive, groups called Stx1

and Stx2. A single EHEC strain may express Stx1 only, Stx2 only, or both toxins or even

multiple forms of Stx2. Stx1 from EHEC is identical to Shiga toxin from Shigella dysenteriae

type I (Kaper et al. 2004).

EHEC is an emerging pathogen that has stimulated worldwide interest in several large food-

borne outbreaks. A wide variety of sources have been implicated in the EHEC transmission,

including beef, unpasteurized milk, fruit juice, and contaminated drinking water (Kaper et al.

2004). EHEC belongs to different serotypes or serogroups, which are useful for diagnostic

and epidemiological studies. The most notorious serogroups associated with EHEC is

O157:H7, which has caused several large outbreaks of the disease, mainly in North America,

Europe, and Japan (Ezawa et al. 2004).

Enteroinvasive E. coli (EIEC)

Like most enteropathogens, these bacteria may also be important pathogens in developing

countries where sanitation and hygiene levels have deteriorated. EIEC strains are

biochemically, genetically, and pathogenically closely related to Shigella spp., but produce

less severe diarrheal disease, compared to the dysentery caused by Shigella dysenteriae type

1 (Nataro & Karper 1998). The precise pathogenetic scheme of EIEC has yet to be

elucidated. However, pathogenesis studies of EIEC suggest that its pathogenic features are

virtually those of Shigella spp. (Sansonetti 1992). EIEC invades the colonic epithelial cell,

thereby inducing an inflammation and mucosal ulceration, leading to the release of blood and

mucus in the stool (Hart et al. 1993), similar to bacillary dysentery.

Although EIEC is the prototype of invasive bacteria, that are unable to penetrate enterocytes

via their luminal aspect. Instead, it passes through the M cells, which are antigen-sampling

cells that are a major constituent of the specialized epithelium overlying the lymphoid

follicles in the small and large intestine (Sansonetti 1992). The ability of EIEC to

penetrate, survive, and multiply within the colonic enterocytes is part of rearrangements of

the cell cytoskeleton, leading to disturbance and engulfment of the bacterium within a

vacuole, a process which is encoded by a cluster of genes carried on a large plasmid .

Afterwards, bacteria move through the cytoplasm and extent into adjacent epithelial cells

without being exposed to the external surroundings. Infection results in a shigellosis-like

syndrome in which patients exhibit abdominal pain, nausea, vomiting, fatigue, mucus, and

bloody stools, but many cases show signs of watery diarrhea that is indistinguishable from

that due to infection by other E. coli pathotypes (Nataro & Karper 1998).

Diffusely adherent E. coli (DAEC)

DAEC is a category of DEC that produces the diffuse adherence on Hep-2 cells (Nataro et

al. 1998) similar to those EAEC strains. However, little is know about the pathogenesis of

DAEC, but cells with a surface with fimbriae that mediate diffuse adherence have been

cloned and characterized (Kaper et al. 2004). Few epidemiological and clinical studies have

been carried out to adequately describe the epidemiology and clinical aspect of diarrhea

caused by DAEC. It was suggested that DAEC may cause disease in immunologically naïve

or malnourished infants. Furthermore, DAEC has been associated to diarrhea in infants older

than 1 year of age (Scaletsky et al. 2002).

Reservoirs

Reservoirs of most diarrheagenic E coli strains are humans, animals and contaminated food

or water (Cole 2006).

Transmission

Transmission of most diarrheagenic E coli strains occurs from consumption of food or water

contaminated with human or animal feces (Kaper 2004; Kassenborg 2004). For some

pathotypes, fewer than1000 colony forming units can cause disease; therefore, person-to-

person transmission from infected symptomatic people or asymptomatic carriers can be an

important mechanism for secondary spread (Belongia 1993; Russo 2006).

EHEC: Major modes of transmission include consumption of contaminated foods (especially

undercooked ground beef), exposure to contaminate recreational or drinking water, or contact

with farm and petting zoo animals (Kassenborg 2004). Person-to-person transmission most

often occurs in schools, long-term care institutions, families, and day-care centers (Belongia

1993; Bopp 2003).

EPEC: The major mode of transmission is person-to-person contact. Although outbreaks

among infants in industrialized countries have largely disappeared, atypical EPEC have

caused large outbreaks of diarrheal disease among children and adults in industrialized

countries (Nguyen 2006). Nosocomial outbreaks have been reported and have been

associated with person-to-person transmission as well as transmission via fomites and

contaminated formula or weaning foods (Nataro 1998).

ETEC: Transmission is primarily via consumption of fecally contaminated food or water

(Qadri 2005).

EIEC: Several foodborne outbreaks have been identified (Gordillo 1992). Person-to-person

spread also can occur.

EAEC: Several nosocomial outbreaks have been identified but the mechanisms of

transmission were not clear. Foodborne transmission has been suggested (Nataro 1998).

DAEC: Modes of transmission have not been clarified. Risk factors for infection include:

i. Consumption of contaminated food, especially undercooked ground beef

(Kassenborg 2004).

ii. Drinking or swimming in contaminated water

Laboratory Diagnosis

Diarrheagenic E. coli is diagnosed by serotyping from stool samples, enzyme immunoassay,

or by other molecular methods (Lopez-Saucedo 2003).

Diagnostic Tests

These pathotypes are identified by detection of their respective virulence-associated factors

(toxins, adherence, or invasiveness). Techniques for testing include bioassays (eg, cell

culture), immunologic assays (eg, immunoblotting or EIA), and DNA assays (eg, PCR,

colony blots).

Methods for identification of ETEC, EPEC, EIEC, DAEC, EHEC and EAEC generally are

available only in reference laboratories or research settings.

Diagnosis of diarrheagenic E coli is difficult for most clinical laboratories, because of the

difficulty in differentiating illness-causing organisms from the usual E. coli strains present in

stool (Voetsch 2004).

Detection of ETEC

Detection of ETEC has long relied on detection of the enterotoxins LT and/or ST. ST was

initially detected in a rabbit ligated ileal loop assay (Evans et al. 1973), but the expense and

lack of standardization caused this test to be replaced by the suckling-mouse assay (Gianella

1976), which became the standard test for the presence of STa for many years. The suckling-

mouse assay entails the measurement of intestinal fluid in infant mice after percutaneous

injection of culture supernatants.

Several immunoassays have been developed for detection of ST, including a

radioimmunoassay and an enzyme-linked immunosorbent assay (ELISA). Both of these tests

correlate well with results of the suckling-mouse assay and require substantially less expertise

(Cryan 1990).

The traditional bioassay for detection of LT involves the use of cell culture, either the Y1

adrenal cell assay or the Chinese hamster ovary (CHO) cell assay. In the Y1 assay, ETEC

culture supernatants are added to Y1 cells and the cells are examined for rounding. In the

CHO cell assay, LT will cause elongation of the CHO cells (Donta et al. 1974).

Immunologic assays are easier to implement in clinical laboratories and include the

traditional Biken test as well as newer immunologic methods such as ELISA, latex

agglutination and two commercially available tests, the reversed passive latex agglutination

test and the staphylococcal coagglutination test. Both of the commercially available tests are

reliable and easy to perform (Honda et al. 1981).

ETEC strains were among the first pathogenic microorganisms for which molecular

diagnostic techniques were developed. As early as 1982, DNA probes were found to be

useful in the detection of LT- and ST-encoding genes in stool and environmental samples.

Since that time, several advances in ETEC detection have been made, but genetic techniques

continue to attract the most attention and use. It should be stressed that there is no perfect test

for ETEC: detection of colonization factors is impractical because of their great number and

heterogeneity; detection of LT and ST defines an ETEC isolate, yet many such isolates will

express colonization factors specific for animals and thus lack human pathogenicity (Moseley

et al. 1981).

Several PCR assays for ETEC are quite sensitive and specific when used directly on clinical

samples or on isolated bacterial colonies. A useful adaptation of PCR is the �multiplex� PCR

assay, in which several PCR primers are combined with the aim of detecting one of several

different diarrheagenic E. coli pathotypes in a single reaction. After multiplex PCR, various

reaction products can usually be differentiated by product size, but a second detection step

(e.g., non-isotopic probe hybridization) is generally performed to identify the respective PCR

products definitively (Lang et a . 1995).

Detection of EPEC

There are two approaches to the detection of EPEC in the laboratory: phenotypic and

genotypic. The phenotypic approach requires the use of cell cultures and fluorescence

microscopy, and the genotypic method requires the use of DNA hybridization or PCR.

Phenotypic tests:

The A/E (attaching and effacing) phenotype can be identified by using cultured HEp-2 or

HeLa cells and the mushroom toxin phalloidin conjugated to rhodamine (available from

Sigma, Molecular Probes,) as described by Knutton and associates (2002).

The A/E phenotype can also be identified by electron microscopic examination of intestinal

biopsy specimens or of cultured epithelial cells incubated with EPEC.

There are a number of phenotypic tests to determine the other crucial characteristic of EPEC,

the lack of Stx expression. An excellent phenotypic marker for the presence of the EAF

(EPEC adhesive factor) plasmid is localized adherence on HEp-2 or HeLa cells. An ELISA

for the detection of EAF-positive EPEC, based on an antiserum raised against an EAF

plasmid containing strain and absorbed against a plasmid-cured strain (Albert et al. 1991).

There are no readily available immunoassays to detect the A/E pattern.

Genotypic tests:

DNA probes and PCR primers have been developed and evaluated for the three major

characteristics of EPEC: A/E, EAF plasmid, and lack of Shiga toxin.

Typical EPEC strains would possess the eae gene for A/E and the EAF probe or bfp

sequences, indicating the presence of the EAF plasmid. Atypical EPEC strains would possess

the eae gene only without the EAF plasmid.

(a) eae gene: Possession of eae sequences correlates with possession of the 35-kb LEE

pathogenicity island encoding A/E (McDaniel et al.1995). No exceptions to this

generalization have been reported, and so there is no need to test for other sequences in the

LEE such as espB and sep unless specific strain differences are sought for molecular

epidemiology purposes (Ebel et al. 1996).

A variety of eae primers for detecting this gene by PCR have been tested. As reported by

Gannon and associates (1997), primer pairs for the conserved 59 region amplified fragments

from all eae-positive strains whereas primers from the 39 end were specific for certain

serotype.

(b) EAF plasmid: The 1-kb EAF fragment probe originally described by Nataro and

associates (1998) is from a region of the EAF plasmid with an unknown function. This probe

may be used with non radioactive labeling techniques. A sensitive and specific

oligonucleotide probe consisting of 21 bases of the EAF fragment was developed by Jerse

and associates (1991). Franke and associates (1989) developed a PCR primer pair to amplify

a 397-bp region of the EAF probe.

The cloning of the bfpA gene allowed the development of DNA probes for specific plasmid-

encoded virulence factors. Giro´n and associates (1997) reported an 850-bp bfpA fragment

probe that was slightly more sensitive than the EAF probe; among EPEC strains exhibiting

localized adherence to HEp-2 cells, the bfpA and EAF probes hybridized with 99 and 96% of

the strains, respectively.

Detection of EHEC

There are three major categories of methods used to diagnose EHEC infections.

These are (i) isolation of E. coli O157 strains from stool samples, (ii) detection of Stx-

producing organisms or fecal Stx, and (iii) detection of elevated antibody levels to O157 LPS

or other EHEC antigens in serum.

Detailed protocols for many of these methods have been reviewed by Smith and Scotland

(Smith and Scotland 1993).

There are no common biochemical characteristics that are associated with the great majority

of EHEC serotypes. However, there are some biochemical characteristics of E. coli O157:H7

that have been exploited in the isolation and identification of this serotype. An important

characteristic is that O157:H7 strains do not ferment D-sorbitol rapidly, in contrast to about

75 to 94% of other E. coli strains. E. coli O157 strains also do not ferment rhamnose on agar

plates, whereas 60% of non-sorbitol-fermenting E. coli belonging to other serogroups ferment

rhamnose (Smith and Scotland 1993).

Culture techniques:

The agar medium most commonly used for the isolation of E. coli O157:H7 is SMAC agar

which is available from multiple commercial sources. This medium contains 1% sorbitol in

place of lactose in the standard MacConkey medium. Sorbitol-nonfermenting colonies,

indicative of E. coli O157:H7, are colorless on this medium. Multiple sorbitol-nonfermenting

colonies (at least 3 and up to 10) should be selected for testing as potential E. coli O157.

SMAC agar is not generally useful for Stx-producing E. coli strains of serotypes other than

O157:H7 because there is no known genetic linkage between Stx production and sorbitol

fermentation (Smith and Scotland 1993).

Immunoassays:

Most of the immunological assays currently available for EHEC detect either O and H

antigens or Stx. The available immunoassays for the O and H antigens are limited largely to

detecting EHEC expressing O157 LPS and the H7 flagellar antigen, whereas toxin

immunoassays are suitable for detecting both O157 and non-O157 serogroups of EHEC.

(i) O and H antigens: Colonies can be screened for the O157 LPS directly from SMAC agar

or after subculture, whereas screening for the H antigen may require passage through motility

medium before testing. Antisera to the O157 LPS and the H7 antigen have been incorporated

into a variety of diagnostic kits involving ELISA, latex reagents, colloidal gold-labeled

antibody, and other formats are available from numerous commercial sources. More

specialized reagents in which antibodies to O157 LPS are already conjugated to fluorescein,

peroxidase, or phosphatase are also available (Sowers et al. 1996).

Commercially available ELISA kits to detect E. coli O157 antigen directly in stool samples

offer testing times of less than 1 hour. These kits are accurate and easy to use in clinical

laboratory settings. Compared to a method involving colony sweeps and immunofluorescence

microscopy, this ELISA had a sensitivity of 91.2% and a specificity of 99.5%. Positive

results from this ELISA should be considered presumptive and should be confirmed by

culture, Stx tests, or PCR (Park et al . 1996).

(ii) Shiga toxins: Numerous immunological assays using various formats have been

developed for the detection of Stx and Stx-producing E. coli. These tests generally take less

than 1 h to complete and have the most potential to be adopted by clinical laboratories for

detection of non- O157:H7 EHEC (Nataro & Karper 1998).

An ELISA system to detect Stx1 and Stx2, which uses antibodies against Stx1 and Stx2 to

capture the antigen, has recently been developed by LMD Laboratories. Another test using a

Gb3 receptor capture system is the VeroTest (Nataro & Karper 1998).

DNA probes and PCR:

Most DNA probes and PCR techniques for EHEC are directed toward the detection of genes

encoding Stx. Frequent loss of stx genes upon subculture can also occur. For these reasons,

probes and PCR techniques for additional EHEC virulence factors can often provide crucial

information.

(i) Detection of stx genes: DNA fragment probes have been used in research studies for

nearly 10 years to detect EHEC. The use of fragment probes requires two different probes,

one for stx1 and one for stx2. With synthetic oligonucleotide probes, at least two probes are

usually used for stx1 and stx2 and additional probes specific for stx2c and stx2e are required

to detect strains containing these genes (Thomas et al. 1993).

The PCR technique has been extensively used to detect stx genes either in stx-only techniques

or in multiplex PCR techniques incorporating primers for eae, ehx, uidA, or fliC (Schmidt et

al. 1995).

(ii) Detection of eae genes: The same eae probes used to identify EPEC can also be used to

identify EHEC if they are derived from the highly conserved 59 end of the gene. The 1-kb

fragment probe described by Jerse and associates (1987) has been shown to be highly

sensitive and specific in many studies. Primers for eae have been combined with primers for

stx1 and stx2 in multiplex PCRs.

(iii) Detection of the pO157 plasmid/hemolysin gene: The pO157 plasmid is present in

nearly all O157:H7 strains and many other EHEC strains. A 3.4-kb fragment probe

empirically derived from this plasmid (called CVD419) was reported by Levine and

associates (1985). This probe has been extensively used to identify and characterize EHEC

strains despite the unknown function of the sequences contained on this fragment. This

fragment was subsequently shown to contain the ehxA (hlyA) genes encoding the EHEC

hemolysin. Oligonucleotide probes and PCR techniques to detect genes encoding the

hemolysin on the pO157 plasmid have also been developed (Thomas et al. 1993).

(iv) Detection of other genes: PCR techniques to detect the gene (fliC) encoding the H7

antigen have been developed. The fliC primer pair has also been combined with primers for

stx and eae in a multiplex PCR to allow the specific identification of E. coli O157:H7,

O157:NM, and other EHEC strains (Gannon et al. 1997).

Serodiagnosis:

Detection of a serum immune response is not usually used for the diagnosis of infections due

to other diarrheagenic E. coli strains, but serodiagnostic techniques can provide valuable

diagnostic information for EHEC infections, particularly since many cases of HUS are not

recognized until after fecal shedding of the organism has ceased. Unfortunately, there is no

single antigen that is ideal for use in serodiagnostic assays. Stx represents an obvious choice

for an important antigen produced in vivo (Nataro & Karper 1998).

Detection of EAEC

There are two major categories of methods used to diagnose EAEC infections.

1) Cell culture:

EAEC infection is diagnosed definitively by the isolation of E. coli from the stools and the

demonstration of the AA pattern in the HEp-2 assay (Fang et al. 1995).The HEp-2 assay

remains the gold standard for detection of EAEC. The presence of bacterial clusters in a

stacked-brick configuration should be used to identify EAEC strains.

The technique as first described by Cravioto and associates (1990) (i.e., a single 3-h

incubation of bacteria with cells, without a change in medium during the course of the assay)

is best able to discriminate the three patterns (AA, DA, and LA). It should also be stressed

that AAF adhesins are maximally expressed in static Luria broth cultures at 37°C.

ii) Molecular methods :

DNA probe: Baudry and associates (1990) selected a 1.0-kb plasmid-derived Sau3a fragment

that hybridized with a fragment of similar size from the 65-MDa plasmid of other strain. In

an evaluation of this fragment as a diagnostic probe, 56 of 63 (89%) EAEC strains (by HEp-2

assay) were positive with the EAEC probe by colony blot hybridization.

A PCR with oligonucleotide primers derived from the probe sequence has also been

developed. The sensitivity and specificity of the EAEC PCR are similar to those of the AA

probe (Schmidt et al. 1995).

Other tests for EAEC:

Several methods other than the HEp-2 and DNA probe assays have been described. Albert

and associates (1991) have reported that EAEC probe-positive organisms display an unusual

pellicle formation in Mueller-Hinton broth. Similarly, when EAEC strains are grown in

polystyrene culture tubes or dishes at 37°C overnight without shaking, a bacterial film is

produced on the polystyrene surface and is easily visualized with Giemsa stain (Nataro et al.

1985).

Detection of EIEC EIEC strains can be difficult to distinguish from Shigella spp. and from other E. coli strains,

including nonpathogenic strains. In general, identification of EIEC entails demonstrating that

the organism possesses the biochemical profile of E. coli, yet with the genotypic or

phenotypic characteristics of Shigella spp.

The classical phenotypic assay for EIEC identification is the Sereny (guinea pig

keratoconjunctivitis) test, which correlates with the ability of the strain to invade epithelial

cells and spread from cell to cell .The ability to form plaques in a HeLa cell monolayer also

correlates with these virulence characteristics (Menard et al. 1994).

Two polynucleotide probe for the detection of EIEC have been described. Probe ial is a 2.5-

kb fragment isolated from an EIEC strain. This probe is virtually 100% sensitive and specific

for EIEC strains that have retained their virulence. A 21-base oligonucleotide derived from

ial is identical in sensitivity and specificity to the polynucleotide probe (Small et al. 1986).

A PCR assay with primers derived from ial was able to detect as few as 10 CFU of S. flexneri

in stool without enrichment of the sample; this compares with 1,000 CFU detectable by DNA

probe. The ial PCR is also effective in a multiplex PCR system to identify EIEC strains

simultaneously with other E. coli categories (Frankel et al. 1989).

Pal and associates (1997) have developed an ELISA to detect the ipaC gene, which is

contained on the plasmid of EIEC. An advantage of this assay over other methods is that it

does not require costly or highly sophisticated equipment.

Detection of DAEC

DAEC strains are defined by the presence of the DA pattern in the HEp-2 adherence assay

(Schmidt et al. 1995). A 700-bp (base pair) polynucleotide fragment derived from the daaC

gene has been used as a DAEC DNA probe; daaC encodes a molecular fragment necessary

for expression of the F1845 fimbriae. Approximately 75% of DAEC strains from around the

world are positive with this F1845 gene probe. However, due to the genetic relatedness of

F1845 to other members of the family of adhesins, false-positive reactions with the DA probe

may occur (Nataro et al. 1996).

No PCR assay has yet been described to identify DAEC.

Antimicrobial Susceptibility

Antimicrobial resistance in E coli has been reported worldwide (Kuntaman 2005; Oteo 2005),

but pathogen occurrence and susceptibility profiles vary region to region (von Baum 2005).

In developing countries resistance levels are usually high for broad-spectrum penicillins and

trimethoprim and low for third-generation cephalosporins and nitrofurantoin (von Baum

2005).The emerging resistance to fluoroquinolones (Kuntaman 2005) and the production of

extended-spectrum beta-lactamases by multidrug resistant E coli strains (Oteo 2005) has

prompted much concern in the developing world, and has implications for developed nations

(Paton 2006). In a study of 3,174 healthy children in Bolivia and Peru, examination of fecal

carriage of drug-resistant E coli was high. Resistance rates were as follows: for ampicilin,

95%; trimethoprim-sulfamethoxazole, 94%; tetracycline, 93%; streptomycin, 82%; and

chloramphenical, 70%. Lower resistance rates were observed for nalidixic acid (35%),

kanamycin (28%), gentamicin (21%), and ciprofloxacin (18%) (Bartoloni 2006).

Treatment

Treatment is usually supportive. Patients who are dehydrated should receive oral

administration of solutions with electrolytes .Children who have inflammatory or bloody

diarrhea should not be given antimotility agents. Patients who have no laboratory evidence of

hemolysis, thrombocytopenia, or nephropathy 3 days after diarrhea resolves have a low risk

of HUS. Those who have hemorrhagic colitis should have careful follow-up (complete blood

cell count with smear, blood urea nitrogen concentration and creatinine levels) to detect

changes that suggest HUS (CDC 2004).

Antibiotic Therapy

Persons with travelers' diarrhea who develop three or more loose stools in an 8-hour period

(especially if associated with nausea, vomiting, abdominal cramps, fever, or blood in stools)

may benefit from antimicrobial therapy. Antibiotics usually are given for 3-5 days. Currently,

fluoroquinolones are the drugs of choice. Commonly prescribed regimens are 500 mg of

ciprofloxacin twice a day or 400 mg of norfloxacin twice a day for 3-5 days. Trimethoprim-

sulfamethoxazole and doxycycline are no longer recommended because of the high level of

resistance to these agents.. Patients suspected of having systemic infection should receive

parenteral antimicrobial therapy (CDC 2005).

Vaccines

No vaccines have been approved, but some vaccine candidates have advanced to clinical

trials and other experimental treatments are being developed.

Infection Control Recommendations

Patients with E coli infections should be managed with Standard Precautions. According to

CDC and the Hospital Infection Control Practices Advisory Committee (HICPAC), Contact

Precautions should be added when caring for diapered or incontinent children younger than

age 6 for the duration of illness (CDC/HICPAC 1996). Place the patient in a private room

or if a private room is not available, place in a room with a patient who has an active

infection with the same pathogen. At least 3 feet of spatial separation should be maintained

between the infected patient and other patients and visitors.

Gloves should be worn when entering the room and removed before leaving the room; hands

should be washed with an antimicrobial agent or waterless hand washing agent immediately

after removing gloves, and clean hands should not touch potentially contaminated items or

environmental surfaces. Gowns should be worn when entering the room if clothing will have

substantial contact with the patient, environmental surfaces, or items in the room; the gown

should be removed before leaving the patient's environment.

Patient transport should be limited to essential purposes only; if the patient is transported out

of the room, precautions should be maintained.

MATERIALS AND METHODS

Type of study:

Descriptive type cross sectional study.

Place of study:

Department of Microbiology, Mymensingh Medical College, Mymensingh.

Period of study:

The study was carried out from January 2011 to December 2011.

Subjects/ cases:

All patients with acute diarrhea irrespective of age and sex admitted in Mymensingh Medical

College Hospital and S.K hospital, Mymensingh were included in this study. Informed

consents were taken from each patient before his/her entry into the study. Information about

children and their consent were taken from their parents/guardians.

Case selection: Children, adult (male and female), with following criteria �

Inclusion criteria:

1) At least 3 times passage of loose stool or watery stool within 24 hours.

2) At least one time passage of bloody stool within 24 hours.

Exclusion criteria:

1) Chronic diarrhea (diarrhea more than 14 days).

Sample or specimen:

Stools were collected as specimen.

Method of collection:

Stool samples (10 mL of liquid or 10 gm of loose stool) were collected in a clean, leak-

proof, wide-mouth container (Cheesbrough 2010).

Sampling technique:

Non- probability purposive type of sampling.

Sample size:

Sample size was calculated by the following formula:

2

2

d

pqZn

n= Desired sample size

p= Prevalence of the disease/problem in community.

q= (1-p)

d= Degree of accuracy (it is, 0.05),

z= Confidence interval (usually we take 95%Ci, in which z =1.96)

According to the prevalence of diarrheagenic Escherichia coli in Bangladesh (34%)

minimum sample size is 344. But in our study sample size is 300 due to limitation of

resources and time.

Data collection instrument:

All relevant history, clinical findings and laboratory records of every case were

systematically recorded in a pre designed data sheet (appendix-I) for subsequent analysis by

computer programme SPSS version 16.0.

Laboratory procedure:

Culture of stool specimen (Isolation of bacteria):

All samples were inoculated into Mac Conkey�s agar (MCA), Salmonella-shegila agar and

Tri-citrate bile sugar media within 10 hours of collection and were incubated at 370C for 24

hours aerobically (Cheesbrough 2010).

Identification of bacteria:

The isolates were identified on the basis of colony morphology, Gram staining, motility and

following biochemical tests.

Grams staining: A drop of distilled water was taken on the centre of a clear glass slide and a

colony was emulsified by a sterilized inoculating loop to make a thin smear. A very thin

smear was made by spreading specimen uniformly. This smear was fixed by passing it over

the flame for two or three times. Smear was covered with crystal violet stain for 30 -60

seconds. Then stain was washed with distilled water and cover with Lugol�s iodine for 30-60-

seconds. Again stain was washed with distilled water and decolourize with acetone alcohol

and wash with distilled water .The back of the slide was wiped and place it in a draining rack,

for the smear to air dry .Test specimen was examined and compared with positive and

negative control under microscope (Cheesbrough 2011).

Motility test: Semisolid agar was used for this purpose. The organism was inoculated by

stabbing a straight wire carrying the inoculam once vertically in to the centre of the agar butt.

After over night inoculation motility was shown by a spreading turbidity from the stab line

or turbidity throughout the medium (Cheesbrough 2010).

Triple sugar iron agar (TSI): This medium was used for initial identification of gram

negative bacilli, particularly member of the Enterobacteriaecae. Three primary characteristics

of a bacterium detect by this medium include ability to produce gas, the production of

hydrogen sulfide gas and the ability to ferment carbohydrate (lactose, sucrose, glucose).To

inoculate a slope and butt medium, a few colonies from a pure culture were picked onto a

straight wire and inoculated to the medium by streaking the surface of the slant slope in a

zigzag pattern and stabbing the butt of the tube all the way to the bottom . This tube was

inoculated at 350C for overnight to observe the colour change of the slant and butt for

differentiation of Enterobacteriaecae ( Cheesbrough 2010).

Indole test: Indole production was tested for some bacteria, which has the ability to degrade

tryptophan to indole. Indole production was detected by Kavac�s reagent (4-dimethyl amino

benzaldehyde, isoamyl alcohol, hydrochloric acid). The test organism was cultured in

peptone broth which contains tryptophan. This broth was inoculated at 350C for overnight

and Kavac�s reagent was added. Development of red colour surface in the broth within 10

minutes indicates positive test (Cheesbrough 2010).

Citrate utilization test: Simon�s citrate agar medium was used for differentiating the

intestinal bacteria and other organism on the basis of citrate utilization .A very light inoculam

was picked with a straight wire and the organism was inoculated to the surface of Semmon�s

citrate agar slant. This tube was inoculated at 350C for overnight to observe the change of

color from light green to blue of the slant (Cheesbrough 2010).

Oxidase test: This test was used for differentiating the intestinal bacteria and other

organism on the basis of oxidase production. A piece of filter paper was placed in a clean

petridish and 2 or 3 drops of freshly prepared oxidase reagent (tetramethyl-p-phenylene

diamine dihydrochloride 10gm/l) was added. With a sterile glass rod 1 or 2 colonies of the

test organisms was taken and rubbed on the filter paper. Development of blue purple colour

within 10 seconds indicates positive test (Cheesbrough 2010).

(The interpretation of the biochemical tests is shown in tabulated form in appendix IV.)

Isolates of Escherichia coli were identified on the basis of pink colour , rounded colonies of

1 � 4 mm diameter in MacConkey�s agar. On Gram stained smear from colonies, those

appeared as Gram negative bacilli. Biochemically isolates were motile, Indole positive,

Citrate negative and Oxidase negative. On TSI, both butt and slunt were found as acidic

(yellow) with production of gas.

Maintenance and preservation of culture strains:

Organisms grown in appropriate media for 18 hours were preserved in a nutrient agar slant at

2-80 C in a refrigerator and these cultures were used within two weeks for laboratory works.

For long term preservation, selected and identified bacterial strains were stored in brain heart

infusion broth with 20% glycerol and stored frozen without significant loss of validity at -200

C until further study (Cheesbrough 2010).

Multiplex PCR for detecting different diarrheagenic Escherichia coli

strains:

Steps:

A) DNA extraction from bacteria isolates.

B) DNA amplification in thermal cycler.

C) Gel electrophoresis and Visualization under UV lights.

A) DNA extraction from bacteria isolates:

DNA of E. coli was extracted from the sweep of few colonies grown on Mac Conkey�s Agar

plates by boiling method.

Steps:

1. One loopful of E. coli from agar plates was suspended in 100 ml of sterile deionized water

in an eppendorf tube and a bacterial suspension was made by using vortex.

2. The bacterial suspension was boiled at 1000C for 10 minutes and centrifuged at 10,000 x g

for 1 min.

3. The supernatant was used as DNA template for PCR. (Nessa et al. 2007)

B) DNA amplification in thermal cycler:

Preparation of reaction mixture:

A total volume of 50 µl master mixture was prepared according to the following composition

(Toma et al. 2003).

Composition of master mixture for each reaction:

10x buffer 5 µl

dNTP (0.2mM each) 4 µl

Primer mixture (30 p mol concentration of each) 3 µl

Taq polymerase ( 2.5 U) 0.5µl

Nuclease free water 36.5µl

DNA template (sample) 1 µl

50 µl

Multiplex PCR was designed for the detection of target genes: eae for enteropathogenic E.

coli (EPEC), eae and stx for enterohemorrhagic E. coli (EHEC), ipaH for enteroinvasive E.

coli (EIEC), CVD432, aspU and aggR for enteroaggregative E. coli (EAEC) and elt and est

for enterotoxigenic E. coli (ETEC).

PCR primers used for detecting different Diarrheagenic Escherichia coli

strains:

Designation - Sequence (5 to 3) - Target gene - Amplicon size (bp) SK1 CCC GAA TTC GGC ACA AGC ATA AGC eae 881 SK2 CCC GGA TCC GTC TCG CCA GTA TTC G VTcom-u GAG CGA AAT AAT TTA TAT GTG stx 518 VTcom-d TGA TGA TGG CAA TTC AGT AT AL65 TTA ATA GCA CCC GGT ACA AGC AGG est 147 AL125 CCT GAC TCT TCA AAA GAG AAA ATT AC LTL TCT CTA TGT GCA TAC GGA GC elt 322 LTR CCA TAC TGA TTG CCG CAA T

ipaIII GTT CCT TGA CCG CCT TTC CGA TAC CGT C ipaH 619 ipaIV GCC GGT CAG CCA CCC TCT GAG AGT AC aggRks1 GTA TAC ACA AAA GAA GGA AGC aggR 254 aggRkas2 ACA GAA TCG TCA GCA TCA GC Eaggfp AGA CTC TGG CGA AAG ACT GTA TC CVD432 194 Eaggbp ATG GCT GTC TGT AAT AGA TGA GAA C spU-3 GCC TTT GCG GGT GGT AGC GG aspU 282 aspU-2 AAC CCA TTC GGT TAG AGC AC Amplification: The prepared PCR tubes with master mixture were placed in the eppendrof thermal cycler

(Eppendrof, Germany). Amplification was carried out according to the following thermal and

cycling condition:

95ºC (Initialization) 5 minutes,

95ºC (Denaturation) 1 minute

56ºC (Annealing) 1 minute 30 cycles

72ºC (Extension) 1 minute

72ºC (Final extension) 10 minutes ( Toma et al. 2003)

C) Gel electrophoresis and Visualization under UV lights:

Agarose gel electrophoresis:

The PCR products were analyzed after electrophoresis in 1.0% agarose gel to detect specific

amplified product by comparing with standard molecular weight marker (Toma et al. 2003).

Preparation of agarose gel:

One percent agarose gel was prepared by melting 2.0 gm agarose in 200 ml of diluted TBE

Buffer(Appendix iv) using a microwave woven. The melted agarose was allowed to cool to

about 50ºC and 20 µl ethidium bromide was mixed and shacked and was poured into gel tray

and combs were placed. After solidification of the gel, the comb was removed. During

electrophoresis, the gel was placed in a Horizontal electrophoresis apparatus containing TBE

buffer and ethidium bromide (Toma et al. 2003).

Loading and electrophoresis of the sample:

Five µl of amplified PCR product was mixed with 2.0 µl of loading buffer. The mixture was

slowly loaded into the well using disposable micropipette tips. Hundred bp molecular weight

marker was loaded in one well to determine the size of the amplified PCR products.

Electrophoresis was carried out at 100 volts for 35 minutes (Toma et al., 2003).

Visualization of the gel:

The amplified products of the study samples were visualized by trans-illuminator. And the

gel was photographed by a digital camera and transferred data to computer for further

documentation (Toma et al. 2003).

Antimicrobial susceptibility testing by disc diffusion method:

All the isolates were put into antibiotic susceptibility test by Kirby-Bauer disc diffusion

technique (Bauer et al., 1966) as per recommendation of CLSI, 2007. Panel of antibiotics

were used. All tests were performed on Muller-Hinton agar (appendix-II). The surface was

lightly and uniformly inoculated by cotton swab. Prior to inoculation, the swab stick was

dipped into bacterial suspension having visually equivalent turbidity to 0.5 McFarland

standards (Appendix-II).The swab stick was then took out and squeezed on the wall of the

test tube to discard extra suspension. Inoculated plates were incubated at 37 0C for 24 hours.

On the next day, plates were read by taking measurement of zone of inhibition. Results were

recorded and graded as Resistant (R) and Sensitive (S) according to the reference zone of

inhibition of particular antibiotic (CLSI, 2007). Known control strain ATCC, No. 25922 and

ATCC No. 25923 were used for quality control.

Antimicrobial agents used

A total of 10 antimicrobial agents were used for determining antibiogram of isolated

organisms according to Gram negative panel recommended by CLSI, 2007. Antibiotics:

Amoxycillin, Cephalexin, Ciprofloxacin, Gentamicin, Amikacin, Nalidixic acid, Ceftriazone,

Cotrimoxazole, Nitrifurantioin and Tetracyclin.

Detection of other enteropathogens :

Other enteropathgens were detected by standard laboratory methods and rotavirus were

detected by PACE (Appendix VI).

Results

A total of 300 stool samples were collected during the study period from Mymensingh

Medical College Hospital and S.K hospital to detect Diarrheagenic E. coli and other

enteropathogens.

The age and sex distribution of the study population is shown in table I, figure 1.1 and figure

1.2. Majority of the cases belonged to <5 years of age (62%) in which 54 (18%) cases were in

the age group <1 year, 132 (44%) cases were in the age group 1-5 years. The rest 114 (38%)

cases were in the age group >5 years. A total of 170 (57%) patients were male and 130

(43%) patients were female.

Of the 300 stool samples examined, Enteropathpgens were detected in 160 (53.5%) cases.

Distribution of enteropathogens in the stool samples is shown in the table II. Single

pathogens were detected in stools of 156 (52%) patients and no pathogens were detected in

140 (46.5%). Multiple pathogens were detected in 4 (1.5%) cases. Rotavirus was the most

common enteropathogen, detected in 82 (27.5%) cases, followed by diarrheagenic

Escherichia coli (DEC) in 54 (18%) cases. Other pathogens detected were Shigella spp. in 8

(2.4%), Salmonella spp. in 5 (1.6%), E. Histolytica in 4 (1.5%) and G. Lamblia in 3 (1.0%)

cases.

Table I. Age and sex distribution of the study population.

Age group (years)

Number of patients (%)

Male Female

<1 54 (18.00) 29 24

1-5 132 (44.00) 56 47

>5-15 76 (25.40) 43 38

>15-45 33 (11.00) 31 21

>45 05 (01.60) 11 0

Total 300 (100) 170 (57%) 130 (43%)

Figure 1.1. Age distribution of the study population.

Figure 1.2 . Sex distribution of the study population.

Table II. Distribution of enteropathogens in the stool samples.

Enteropathogen no. of patients Percentage

Rotavirus 82 27.5

Diarrheagenic Escherichia coli 54 18.0

Salmonella spp. 05 1.6

Shigella spp. 08 2.4

E.Histolytica 04 1.5

G.lamblia 03 1.0

Mixed pathogens 04 1.5

No pathogen identified 140 46.5

Total 300 100

Table III. shows distribution of DEC Strains in the study population. Enterotoxogenic E. coli

(ETEC) was detected in 39 (13%) cases and Enteroaggregative E. coli (EAEC) in 15 (5%)

cases. No Enterohemorrhagic E. coli (EHEC), Enteropathogenic E. coli (EPEC) and

Enteroinvasive E. coli (EIEC) could be detected.

Table IV. shows distribution different genes of ETEC Strains in the study population. Both

(ST and LT) genes of ETEC were detected in 26 (66.68%) strains, ST gene in 9 (23.07%)

strains and LT gene in 4 (10.25%) strains.

Table V. shows detection rate of Enterotoxogenic Escherichia coli (ETEC) in different age

group of the study population. Highest detection of ETEC 19 (6.34%) were in the age group

1-5 years, followed by age group >5 years 16 (5.33%) and 4 (1.33%) in the age group <1

year.

Table VI. shows detection rate of Enteroaggregative Escherichia coli (EAEC) in different

age group of the study population. Highest detection of EAEC 8 (2.70%) were in

the age group 1-5 years, followed by age group <1 year 5 (1.64%) and 2 (0.66%) in

the age group >5 years.

Table III: Distribution of different diarrheagenic Escherichia coli (DEC)

Strains in the study population (n=300).

Diarrheagenic Escherichia coli Number of cases (%).

ETEC 39 (13)

EAEC 15 (5)

EPEC 0 (0)

EIEC 0 (0)

EHEC 0 (0)

Total 54 (18)

Table IV: Distribution of different genes (ST / LT/ Both) of

Enterotoxogenic Escherichia coli (ETEC) strains in the study population.

Diarrheagenic

Escherichia

coli

Number of

strains

ST

gene (%)

LT

gene (%)

Both

(ST&LT)

genes (%)

ETEC 39 9(23.07) 4(10.25) 26(66.68)

Table V. Detection rate of Enterotoxogenic Escherichia coli (ETEC) in

different age group of the study population (n=300).

Age group

(years) Number of ETEC positive cases (%).

<1 04 (1.33)

1-5 19 (6.34)

>5-15 10 (3.33)

>15-45 06 (2.00)

>45 00 (00)

Table VI. Detection rate of Enteroaggregative Escherichia coli (EAEC) in

different age group of the study population (n=300).

Age group

(years) Number of EAEC positive cases(%).

<1 05 (1.64)

1-5 08 (2.70)

>5-15 02 (0.66)

>15-45 00 (00)

>45 00 (00)

Clinical features of Enterotoxogenic Escherichia coli (ETEC) in the study population are

shown in table VII. Watery diarrhea and vomiting were the chief complaint and were present

in 87% and 46% cases respectively. No cases were found with bloody stool. Fever occurred

in 64% of the infected patients. Clinical assessment for dehydration revealed some and severe

dehydration in 38% and 62% of the cases respectively.

Clinical features of Enteroaggregative Escherichia coli (EAEC) in the study population are

shown in table VII. Watery diarrhea and vomiting were the chief complaint and were present

in 80% and 60% cases respectively. No cases were found with bloody stool. Fever occurred

in 74% of the infected patients. Clinical assessment for dehydration revealed some and severe

dehydration in 20% and 80% of the cases respectively.

Table IX. shows antimicrobial susceptibility patterns of Enterotoxogenic E. Coli (ETEC)

isolated from the stool samples. ETEC isolated from the stool samples were 100% sensitive

to Ceftriaxone, Nitrofurantioin and Amikacin, 94% to Nalidixic acid, 89% to Gentamycin,

83% to Ciprofloxacin, 79% to Cephalexin, 46% to Tetracycline, 39% to Amoxycillin and

31% to Cotrimoxazole.

Table IX. shows antimicrobial susceptibility patterns of Enteroaggregative E. Coli (EAEC)

isolated from the stool samples. EAEC isolated from the stool samples were 100% sensitive

to Ceftriaxone, Nitrofurantioin, Amikacin and Nalidixic acid, 90% to Gentamycin and

Ciprofloxacin, 85% to Cephalexin, 41% to Amoxicillin, 49% to Tetracycline and 31% to

Cotrimoxazole.

Table VII: Clinical features of Enterotoxogenic Escherichia coli (ETEC) in

the study population.

Clinical features ETEC infected Cases (%).

Watery stool 34(87.17)

Loose stool 05(12.83)

Bloody stool 00(00)

Vomiting 18(46.15)

Fever 25(64.10)

No dehydration 00(00)

Some dehydration 15(38.46)

Severe dehydration 24(61.54)

Table VIII: Clinical features of Enteroaggregative Escherichia coli (EAEC)

in the study population.

Clinical features EAEC infected Cases (%).

Watery stool 12(80)

Loose stool 03(20)

Bloody stool 00(00)

Vomiting 09(60)

Fever 11(74)

No dehydration 00(00)

Some dehydration 03(20)

Severe dehydration 12(80)

Table IX: Antibiotic sensitivity patterns of Enterotoxogenic E. coli

(ETEC) isolated from the stool samples (n=39).

Antibiotic Number of ETEC strains sensitivity to

different antibiotics (%).

Ceftriaxone 39(100)

Amikacin 39(100)

Nitrofurantioin 39(100)

Nalidixic acid 36(94)

Gentamycin 34(89)

Ciprofloxacin 32(83)

Cephalexin 30(79)

Tetracycline 18(46)

Amoxycillin 15(39)

Cotrimoxazole 12(31)

Table X: Antibiotic sensitivity patterns of Enteroaggregative E. coli

(EAEC) isolated from the stool samples (n=15).

Antibiotic Number of EAEC strains sensitivity to

different antibiotics (%).

Ceftriaxone 15(100)

Amikacin 15(100)

Nalidixic acid 15(100)

Nitrofurantioin 15(100)

Ciprofloxacin 13(90)

Gentamycin 13(90)

Cephalexin 12(85)

Tetracyclin 7(49)

Amoxycillin 6(41)

Cotrimoxazole 5(31)

Discussion

Acute diarrhea is one of the most common illness and cause of death in young children in

Bangladesh. The present study to detect diarrheagenic Escherichia coli (DEC) is meaningful

in that it provides information about the prevalence of different diarrheagenic E. coli in the

study population.

In the present study, majority of the cases belonged to <5 years of age (62%) in which 54

(18%) cases were in the age group <1 year, 132 (44%) cases were in the age group 1-5 years.

The rest 114 (38%) cases were in the age group >5 years (Table I). Many studies have

reported that diarrhea is more common in children under 5 years of age in both developed and

developing countries (Antikainen et al. 2009). Albert and associates (1995) reported that 53%

of diarrheal patients were below 5 years of age in Bangladesh, which supports the present

study findings. Stoll and associates (1982) reported that most of the diarrheal patients were

children in Bangladesh: 20% were under 1 year of age, 42% between 1 and 5 years, 12%

between 5 and 15, and 26% were 15 and over, which also correlates with the present study

findings. In a recent study from Vietnam by Nguyen and associates (2005), it was found that

diarrhea was more frequent in children less than 5 years of age, which correlates with the

present study findings. Similar findings were reported in other study in Germany (Presterl et

al. 2003).

Out of 300 cases, 170 (57%) were male and 130 (43%) were female. The male and female

ratio was 1.32:1. These findings were similar with the study done by Albert and associates

(1999) where they found the male and female ratio was 1.42:1. Similar findings of male

dominance over female cases were reported in other study in Bangladesh (Strockbine et al

1992). The increase number of male patients over female irrespective of age in the present

study might be due to more exposure of male in the external environment and taking out-door

foods which has more chance to be contaminated by diarrheal pathogens (Nataro & Karper

1998).

Of the 300 stool samples examined, Enteropathpgens were detected in 160 (53.5%) cases.

Overall, Rotavirus was detected in 82 (27.5%) cases, followed by diarrheagenic Escherichia

coli (DEC) in 54 (18%), Shigella spp. in 8 (2.4%), Salmonella spp. in 5 (1.6%), Entamoeba

Histolytica in 4 (1.5%) and Giardia. Lamblia in 3 (1.0%) cases (Table II). The prevalence of

enteropathogens in the study population was lower compared to previous study carried out in

Bangladesh (ICDDR,B 2002) where they found association of enteropathogens with 81%

diarrheal diseases. In biological research some sorts of variation is not unexpected because it

depends upon some factors like age, seasonality, epidemicity, methods of samples collection

and tools used (Antikainen et al. 2009). Among bacterial pathogens, Campylobacter spp.,

Yersina spp. and Plesiomonas spp. and some recognized viral agents of diarrhea (enteric

adenovirus, Calici virus and astrovirus) were not sought in the present study due to resource

constraints.

In the present study, rotavirus (27.5%, 82/300) was the most predominant cause of acute

diarrhea in the study population. This finding for rotavirus infection was slightly lower than

similar study reported by ICDDR, B (ICDDR, B 2002) and slightly higher than other studies

done in Bangladesh (Paul et al. 2008; Baqui et al. 1992). The lower prevalence than ICDDR,

B might be due to use of different detection method (PAGE) in the present study.

Among the bacterial pathogens, diarrheagenic Escherichia coli (18%, 54/300) was the most

prevalent in the study population. The prevalence of DEC was lower compared to previous

studies carried out in Bangladesh. In 2002, it was reported that DEC was responsible for 34%

of diarrheal diseases in Bangladesh (ICDDR, B 2002). In 1999, Albert and associates (1999)

found that DEC was associated with 30% of diarrheal diseases in Bangladesh. All the above

study findings were higher than the present study findings. Various factors might be

responsible for such a difference. The samples collected were neither directly inoculated in

culture media nor transported by transport media, history of taking antibiotics before sample

collection and seasonality might be important factors to reduce the identification of DEC in

the study population (Stoll et al. 1982).

In Iraq, Sehand and associates (2010) and in Vietnum, Nguyen and associates (2005) found

DEC was responsible for 20% and 22.5% of diarrheal diseases respectively, which correlates

well with the present study findings.

Shigella spp. was found to be the second most bacteria in the present study and the detected

rate was 2.4% (8/300). In previous studies carried out in Bangladesh, Shigella spp. detection

rate varied from 2.6% to 12% in diarrheal patients (Albert et al. 1999; Stoll et al. 1982). The

isolation rate of Shigella spp. was highly variable from country to country which ranges from

1.6% in Ghana to 27% in Jardan (Youssef et al. 2001).

In the present study, the prevalence of Salmonella spp. was found to be 1.6% (5/300) which

was all most similar with the study findings reported by ICDDR, B in 2002 (ICDDR,B 2002).

Albert and associates (1995) found that Salmonella spp. was associated with 1.1% of

diarrheal disease in Bangladesh, which supports the present study results.

Among diarrheagenic parasites E . Histalytica and G. lamblia were found in 1.4% (4/300)

and 1.0% (3/300) cases in the present study respectively. Similar results were reported by

Albert and associates (1999) in Bangladesh. Recently it is reported that the prevalence of

diarrheagenic parasites is decreasing in Bangladesh (ICDDR, B 2003), it may be due to

improvement in sanitation, personal hygiene and safe water supply.

In the present study, among the diarrheagenic Escherichia coli (18%, 54/300),

Enterotoxogenic E. coli (ETEC) was detected in 39 (13%, 39/300) patients and

Enteroaggregative E. coli (EAEC) in 15 (5%, 15/300) patients (table III). No

Enterohemorrhagic E .coli (EHEC), Enteropathogenic E. coli (EPEC) and Enteroinvasive E.

coli (EIEC) could be detected. ETEC was found to be the most prevalent DEC in the study

population. ETEC has been attributed as a common diarrheal pathogen in children in many

developing countries of Asia, Africa and South America (Nguyen et al. 2005). In Bangladesh,

ETEC was reported as a cause of diarrhea with a prevalence of 14.0%, which correlates well

with the present study findings (Qadri et al. 2005). In 2002, it was reported that ETEC was

responsible for 12.7% of diarrheal diseases in Bangladesh, which was almost similar with the

present study findings (ICDDR,B 2002). Albert and associates (1999) showed that ETEC was

associated with 15.8% of diarrheal disease in Bangladesh, which supports the present study

results. Giron and associates (1995) and Stoll and associates (1982) found ETEC was

responsible for 19% and 20% of diarrheal diseases in Bangladesh respectively, which was

slightly higher than the present study findings.

Similar studies with acute diarrhea were performed in different parts of world. In Japan,

different types of diarrheagenic E. coli were the most frequently identified enteropathogens

(Toma et al. 2003). In Iran, Arif and associates (2010) and in Finland, Antikainen and

associates (2009) found ETEC was associated with 10% and 11% of diarrheal diseases

respectively, which correlates well with the present study findings. However in Thailand,

Hein and associates (2008) and in Vietnam, Nguyen and associates (2005) found ETEC was

associated with 5.8% and 4% of diarrheal diseases respectively, which was lower than the

present study findings. This might be due to important regional differences in the prevalence

of different categories of diarrheagenic E. coli (Albert et al. 1995).

EAEC (5%, 15/300) was the second most prevalent DEC in the present study population.

EAEC has been found as a common diarrheal pathogen in children in many developing

countries (Nguyen et al. 2005). In 2002, it was reported that EAEC was responsible for 6.1%

of diarrheal diseases in Bangladesh (ICDDR, B 2002). Albert and associates (1999) found

EAEC was associated with 6.9 % of diarrheal diseases in Bangladesh. In 1992, Faruque and

associates also found EAEC was responsible for 5.9% diarrheal diseases in Bangladesh. All

the above study findings correlate well with the present study findings.

EAEC has been implicated as the etiological agent of diarrhea not only in developing

countries but also as a cause of gastroenteritis outbreaks in some industrialized countries. It

was reported as the cause of a massive outbreak of gastrointestinal illness in school children

in Japan, as the cause of acute diarrhea in children in Brazil, and in other developed countries

(Smith et al.1997; Fang et al. 1995; Schmidt et al. 1995). In Iran, Arif and associates (2010)

foumd that EAEC was associated with 6.7% diarrheal diseases, which correlates with the

present study findings. However Rappelli and associates(2005) reported that EAEC was

responsible for 9.1% diarrheal diseases in Finland, which was higher than the present study

findings. In Vietnam, Nguyen and associates (2005) showed that EAEC was associated with

11% diarrheal diseases which was also higher than the present study findings. This might be

due to important regional differences in the prevalence of different categories of

diarrheagenic E. coli (Alert et al. 1995).

EPEC could not be detected in the present study population. The most important feature of

the diarrheal diseases due to EPEC infection is the remarkable age distribution. EPEC

infection is primarily a disease of infants younger than 1 year of age (Nataro & Karper 1998).

In the present study, only 18% cases were found in the age group below 1 year. Unicomb and

associates (1996) and Nessa and associates (2007) could not detect EPEC from any age group

in Bangladesh. However Baqui and associates (1992) and, Albert and associates (1995) found

EPEC in 8% and 12% cases respectively in Bangladesh.

In the present study, EIEC and EHEC could not be detected in the study population. In many

previous studies carried out in Bangladesh, EIEC and EHEC could not be detected in

diarrheal patients (Strockbine et al 1992; Albert et al. 1995: ICDDR, B 2002; Baqui et al.

1992)). The absence of these strains is not surprising since these pathotypes are not

frequently detected in developing countries of Africa and Asia (Rappelli et al. 2005).

In the present study, ST gene of ETEC was found in majority of cases compared to LT gene.

Out of 39 Enterotoxogenic Escherichia coli, both (ST and LT) genes of ETEC were detected

in 26 (67%) cases, ST gene in 9 (23%) cases and LT gene in 4 (10%) cases (Table IV).

Significant association with diarrhea was seen with ETEC producing both ST and LT and

ETEC producing only ST; ETEC producing only LT was not significantly associated with

diarrhea. This trend agrees with the findings in other studies in Bangladesh (Albert et al.

1995; Qudri et al. 2005). Similar findings were reported in other study in Finland (Antikainen

et al. 2009).

In the present study, highest detection of ETEC 19 (6.34%) were in the age group 1-5 years,

followed by age group >5 years 16 (5.33%) and 4 (1.33%) in the age group <1 year (Table

V). Majority of ETEC (8%) belonged to <5 years of age. It was reported that most of the

diarrheal patients infected with ETEC were <5 years of age in Bangladesh (ICDDR,B 2002).

Qudri and associates (2000) also reported that the most of the diarrheal patients infected with

ETEC were children <5 years of age in Bangladesh. All the above study findings were similar

with the present study findings.

Many studies have reported ETEC as a major agent of traveller�s diarrhoea in adults, and the

leading cause of child diarrhoea in both developed and developing countries (Antikainen et

al. 2009; Parashar et al. 2003). Those findings are correlated with the present study findings

as the high prevalence of ETEC was detected in child and adults in the present study

population.

In the present study, highest detection of EAEC 8 (2.70%) were in the age group 1-5 years,

followed by age group <1 year 5 (1.64%) and 2 (0.66% ) in the age group >5 years (Table 5).

Majority of EAEC (4.5%) belonged to < 5 years of age. Baqui and associates (1992) reported

that the most of the diarrheal patients infected with EAEC were children <5 years of age in

Bangladesh which agrees with the present study findings. Nessa and associates (2007) also

found association of EAEC mostly with diarrheal patients of <5 years of age in Bangladesh.

In a recent study from Japan by Nguyen and associates (2006), it was shown that EAEC was

more frequently associated with diarrhoea in children less than 5 years of age, which

correlates with the present study findings.

In the present study, Watery diarrhea and vomiting were the chief complaint and were present

in 87% and 46% respectively in patients infected with ETEC. No cases were found with

bloody stool. Clinical assessment for dehydration revealed some and severe dehydration in

38% and 62% of the cases respectively. Similar findings were reported in other studies in

Bangladesh (Stoll et al. 1982; Qudri et al. 2005). Jafari and associates (2008) also found

similar findings in Tehran.

In the present study Watery diarrhea and vomiting were the chief complaint and were present

in 80% and 60% respectively in patients infected with EAEC. No cases were found with

bloody stool. Clinical assessment for dehydration revealed some and severe dehydration in

20% and 80% of the cases respectively. Similar findings were reported in other study in

Bangladesh (Stoll et al., 1982; Albert et al. 1995). Presterl and associates (2003) also found

similar findings in Germany.

In the present study, detected ETEC were 100% sensitive to Ceftriaxone, Nitrofurantioin,

Amikacin, 94% sensitive to Nalidixic acid, 89% sensitive to Gentamycin, 83% sensitive to

Ciprofloxacin, 79% sensitive to Cephalexin, 39% sensitive to Amoxycillin, 46% sensitive to

Tetracyclin and 31% sensitive to Cotrimoxazole. Detected EAEC were 100% sensitive to

Ceftriaxone, Nitrofurantioin, Amikacin, Nalidixic acid, 90% sensitive to Gentamycin and

Ciprofloxacin, 85% sensitive to Cephalexin, 41% sensitive to Amoxycillin, 49% sensitive to

Tetracycline and 31% sensitive to Cotrimoxazole. Both ETEC and EAEC isolates exhibited

decreased susceptibility for Amoxycillin, Tetracycline and Cotrimoxazole. This trend agrees

with the findings in other study in Germany (Presterl et al. 2003). kalantar and associates

(2011) found similar type of results that supports the present study findings.

The prevalence of all the five categories of diarrheagenic E. coli was studied previously in

Bangladesh. However, these studies suffered from several deficiencies which included the

following: (i) not all categories of E. coli were sought in a single study, which resulted in

generation of data in a piecemeal fashion and (ii) some researchers relied on phenotype-based

tissue culture assay for aggregative E. coli, whose interpretation is prone to observer variation

(Strockbine et al. 1992; Albert et al. 1995). The present study overcomes all these

deficiencies and shows that multiplex PCR is a suitable method for DEC detection by

amplifying specific genes, where facilities are available.

Conclusion and Recommendations

Analyzing the findings of the present study, diarrheagenic Escherichia coli was found to be

one of the most important cause of acute diarrhea. Detection rate of diarrheagenic

Escherichia coli by multiplex PCR was quite satisfactory. Thereby, Multiplex PCR could be

adapted to detect different Diarrheagenic Escherichia coli in setup like Medical colleges or

tertiary medical facilities.

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PHOTOGRAPH

Vortex Mechine

Dry water bath.

Photograph of Centrifuge Machine

Photograph of Thermal cycler

Photograph of Horizontal Electrophoresis Chamber

Photograph of Documentation

chamber wit

eae gene � 881 bp

Stx gene-518 bp

est gene-147 bp

elt gene-322 bp

ipaH gene-619 bp

aggR gene-254 bp

CVD432 gene-194 bp

aspU gene -282 bp

PCR of Different diarrheagenic Esch coli

322 bp

147 bp

254 bp 600bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

147bp

1500 bp

Photograph of Multiplex PCR showing aggR gene (254 bp) of EAEC in lane 1,

est gene (147 bp) and elt gene (322 bp) of ETEC in lane 4, only est gene (147

bp) of ETEC in lane 14 and ladder marker (100bp) in lane 5 and 12.

Photograph of Mac-Conkey agar medium showing pink colure colonies of E

.coli.

A B C

Photograph of biochemical reaction of E. coli in TSI agar medium.

A- Positive control showing yellow shunt and butt.

B- Test organism showing yellow shunt and but

C- Negative control showing red shunt and butt.

A B C

Photograph of Indole test of E. coli. A- Positive control showing pink colored ring at the top of the broth medium.

B- Test organism showing pink colored ring at the top of the broth medium.

C- Negative control showing no colored ring at the top of the broth medium

A B C Photograph of Citrating utilization test of E . coli. A - Positive control showing no change of colure in the medium.

B- Test organism showing no change of colure in the medium.

C- Negative control showing blue colure in t the medium..

Polyacrylamide gel electrophoresis Chamber

Extracted RNA

Loading In Electrophoresis Chamber

Visualized RNA Segments of Rotavirus Electrophoresis

Staining

Pictorial presentation of major steps of PAGE

APPENDICES Appendix � I

DATA SHEET

Thesis title: �Detection of Different Diarrheagenic Escherichia coli Strains by multiplex PCR�. A. S.L No: Identification number :

B. Particulars of the patients: 1. Name:

2. Age:

3. Sex: Male/Female

4. Address:

Village: P.O: Union: Thaana: Dist:

5. Admitted patients: Ward no: Bed no: Reg.no:

6. Date of admission:

7. Socio-economic condition:

8. Housing condition: city/village/slum area.

9. Latrine: sanitary/non sanitary (kacha/paka/open).

10. Source of drinking water: supply/tube well/pond/River. C) Presenting complains:

a) Duration of diarrhea for : days b) Onset: c) Frequency of motion: e) Nausea/abdominal discomfort/ anorexia/vomiting.

D) On examination:

1. Pulse /min.

2. Anemia:

3. Abdominal tenderness

4. Dehydration

5. Temp:

Past history: History of antibiotic intake: Name of antibiotic: dose & duration: Complication:

LABORATORY FINDINGS:

Specimen: stool.

Microscopic examination:

Pus cells:

E. histolytica: Not found/ Cyst/ Trophozoite

G. lamblia: Not found/ Cyst/ Trophozoite

Culture:

Organism isolated: Culture Finding: On MacConkey agar media: On Salmonella-Shigella agar media:

Gram staining : Gram Positive Cocci/ bacilli.

Gram Negative Cocci / bacilli.

Motility test: Motile/ Nonmotile

Biochemical tests:

i) Triple sugar iron agar:

shunt:

butt:

ii) Indole test : Positive\ Negative

iii) Citrate utilization test: Positive\ Negative

iv)Oxidase test: Positive\ Negative

Antibiotic sensitivity:

Antibiogram of isolates (CLSI, 2007):

S= sensitive, R= Resistant,

PCR result : Signature:

Zone diameter (mm) Antibiotics Disk content

µgm/disc Resistant Sensitive

Result Comments

Amoxycillin 10 ≤ 13 ≥ 17

Tetracycline 30 ≤ 11 ≥ 15

Cotrimoxazole 25 ≤ 10 ≥ 16

Gentamicin 10 ≤ 12 ≥ 15

Ciprofloxacin 5 ≤15 ≥ 21

Nitrofurantoin 300 ≤ 14 ≥ 17

Nalidixic acid 30 ≤13 ≥ 19

Ceftriaxone 30 ≤13 ≥ 21

Amikacin 15 ≤ 13 ≥ 18

Cephalexin 30 ≤12 ≥ 18

Appendix � II

The composition and methods of preparation of different media, chemicals and reagents used

in this study are given below �

Muller Hinton agar medium

Composition:

Ingredients gram/liter Beef dehytrated infusion 300 Casein hydrolysate 17.50 Starch agar 17.00 Agar 17.00

Thirty eight grams of dehydrated Mueller Hinton agar medium was suspended in 1000 ml

cold distilled water and boiled to dissolve the medium completely. The solution was then

sterilized by autoclaving at 1210C and 15 lbs pressure for 15 minutes. The autoclaved media

was stored at 40C

MacConkeys agar medium

Composition: Ingredients gram/liter Peptone 19.0 Lactose 10.0 NaCl 5.0 Na- Deoxycholat 1.0 Neutral Red 0.03 Crystal Violet 0.001 Agar 15.0

Fifty two grams of dehydrated MacConkeys agar medium was suspended in 1000 ml cold

distilled water and boiled to dissolve the medium completely. The solution was then sterilized

by autoclaving at 1210C and 15 lbs pressure for 15 minutes.

Triple sugar iron agar:

Composition:

Ingredients gram/liter Peptic digest of animal tissue 10.00 Casein enzymic hydrolysate 10.00 Yeast extract 3.00

Beef extract 3.00 Lactose 10.00 Sucrose 10.00 Dextrose 1.00 Sodium chloride 5.00

Sixty five grams of dehydrated triple sugar iron agar medium was suspended in 1000 ml cold

distilled water and boiled to dissolve the medium completely. The solution was then sterilized

by autoclaving at 1210C and 15 lbs pressure for 15 minutes.

Simon�s citrate medium

Composition:

Ingredients Dehydrated media 24.2 gram

Distilled water 1000.0 ml

pH- 6.8, Ingredients dissolved, dispense into test tubes autoclaved at 1210C for 15 minutes

and allowed to set as slopes.

MacFarland Standard 0.5

Composition and preparation 1% (V/V) solution of chemically pure 0.36 (N) sulphuric acid

and 1.175% (W/V) solution of chemically pure (0.048M) barium choloride was prepared in

two separate sterile flasks. Than 9.9ml of sulphuric acid and 0.1ml of barium chloride were

added to the clean screw capped test tube and sealed. The barium sulphate suspension

corresponds approximately to Mcfarland standard tube number1 with corresponding cell

density of 3× 108 organism/ ml to made the turbidity standard of cell density to one half of

the Mcfarland standard tube number 1 which correspond to cell density of 1.5 ×108

organism/ml for determination of antibiotic sensitivity by Kirby-Bauer inoculated technique

0.5ml of 1.75% (W/V) barium chloride (Bacl2 2H2) was added to 99.5ml of 1% (V/V)

sulphuric acid (0.36N), mixed well and 5 � 10 ml was distributed in sterile capped test tube

and sealed.

Kovac�s reagent (Collee et al, 1996)

Composition

Ingredients Amyl or isoamyl alcohol 150ml P-Dimethyl-aminobenzaldehyde 10gram Concentrated hydrochloric acid 50ml

Aldehyde was dissolved in alcohol and acid is added slowly.

Method: Medium was inoculated with test organisms and incubated for 48 hours at 370C. 0.5

ml of Kovacs reagent was added and shaken gently. A red colour in the alcohol layer

indicates a positive reaction.

Oxides reagent

To make 10ml

Tetramethyl-p-phenylenedimine(dihydrocholoride) 0.1g

Distilled water 10 ml

Tris-EDTA buffer:

To make 1 litre TE buffer required:

Tris (10mM)������.1.21gm

EDTA (1mM)������0.372gm

Distilled water������1 litre

EDTA will not be soluble until pH reaches 8.0. pH was checked with pH papers and then

stored at 2-8ºC.

6× Disruption solution for PAGE

6% Sodium Dodecyl Sulphate (SDS), 0.6% 2-mercptoethanol and 60 mM Ethylene diamine tetra acitic acid (EDTA).

10% polyacrylamide gel (2ml)

0.75 M tris-HCL buffer (pH 8.8)- 20ml, Acrylamide solution- 13.3 ml, o.1 M EDTA- 0.4 ml, Distilled water- 4.7 ml, 3% amonnium per sulfate- 1.6 ml and TEMED (Tetramethlenediamine)- 70 µl.

Appendix � III

The composition and methods of preparation of different stains, diluents and

chemicals used in this study are given below

Crystal violet Gram stain:

Crystal violet 20g

Ammonium oxalate 9g

Ethanol or methanol, absolute 95ml

Distilled water to 1 liter

Lugol�s iodine solution:

Potassium iodide 20g

Iodine 10g

Distilled water 1 liter

Alcohol fixative solution:

To make 200ml

Ethanol (ethyl alcohol), absolute 180ml

Acetic acid, glacial 10ml

Distilled water 10ml

Immerse the fixative for 20 minutes. Rinse with 95% ethanol and allow the smear to

dry.

Carbol fuchsin:

Basic fuchsin 10g

Ethanol or methanol 100ml

Phenol 50g

Distilled water 1 liter

Appendix � IV

The biochemical results were interpreted as per the following table:

TSI Species Oxidase

Citrate

utilization Indole

slant butt H2S gas

E. coli - - + Y

Acidic

Y

Acidic

- +

Shigella

spp.

- - - R

Alkalne

Y

Acidic

- -

Klebsilla

spp.

- + - Y

Acidic

Y

Acidic

- +

Salmonela

spp.

- + (d) - R

Alkalne

Y

Acidic

+ +(d)

Positive= (+)

Negative= (--)

Yellow=Y

Red=R

D= different strains give different results.

Appendix � V

Equipments for PCR

Centrifuge 5415 R with refrigerator

Vortex Mixer (Digi system VM-2000)

Micropipettee (Company-Eppendrof)

Laminar Flow Cabinet (Company stremline)

Microwave oven for gel preparation

Electric heated dryer (Model Jo2714)

Thermal cycler (Model-Master cycler personal)

Gel apparatus (Major science Minis-300)

Major science Minis UVDI

Appendix � VI

Rotavirus detection by PAGE (Polyacrylamide Gel electrophoresis):

This test is based on the detection of rotavirus double stranded RNA genome which is

separated into 11 segments by electrophoresis through polycrylamide gels.

Steps (Extraction of RNA, electrophoresis and staining):

1. Transfer the 300 µl sample to 1.5 ml tube.

2. Add 60 µl of 6× Disruption solution and vortex.

3. Incubate at 370C for 30 minutes.

5. Centrifuge at 12000 rpm for 1 minute.

6. Transfer supernatant (300 µl) to a new tube.

7. Add 600 µl of ethanol and add 10 µl of 5M NaCl and mix by inverting.

8. Incubate at 800C for 30 minutes.

9. Centrifuge at 12000 rpm for 15 minutes.

10. Discard the supernatant.

11. Dry up to for 15 minutes.

13. Add 10 µl of loading buffer.

14. Subject RNA sample to electrophoresis in a polyacrylamide slab gel (for 4 hours).

15. Stain the Gel with Silver nitrate for 40 minutes.

Silver Staining of the gel

1. Stain the gel with silver nitrate (0.2 gm in 100 ml) for 40 min.. 2. Wash the gel 3 times with distilled water. 3. Stain the gel with NaOH (3 gm in 100ml) and formaldehyde (HCHO) (0.4 ml) for 10 min. 4. Add stop solution (5% acetic acid).

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