Prevalence and Molecular Characterization of...

Prevalence and Molecular Characterization of Diarrheagenic

Escherichia coli in Southern Khyber Pakhtunkhawa, Pakistan

By

Mir Sadiq Shah

Department of Microbiology

Quaid-i-Azam University

Islamabad, Pakistan

2015

Prevalence and Molecular Characterization of Diarrheagenic

Escherichia coli in Southern Khyber Pakhtunkhawa, Pakistan

A thesis

Submitted in the Partial Fulfillment of the

Requirements for the Degree of

DOCTOR OF PHILOSOPHY

IN

MICROBIOLOGY

By

Mir Sadiq Shah

Department of Microbiology

Quaid-i-Azam University

Islamabad, Pakistan

2015

DECLARATION

The material contained in this thesis is my original work and I have not presented any part of this

thesis/work elsewhere for any other degree.

Mir Sadiq Shah

To

My parents

CERTIFICATE

This thesis, submitted by Mr. Mir Sadiq Shah is accepted in its present form by the Department

of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad as

satisfying the thesis requirement for the degree of Doctor of Philosophy (PhD) in Microbiology.

Internal Examiner: _______________________________

(Dr. Fariha Hasan)

External Examiner: _____________________________

External Examiner: ______________________________

Chairperson: ____________________________________

(Dr. Fariha Hasan)

Dated:

CONTENTS

S. No Title Page. No

1. List of Abbreviations i

2. List of Tables iii

3. List of Figures IV

4. Acknowledgements VI

5. Abstract VIII

6. Chapter 1: Introduction 1

7. Chapter 2: Literature Review 19

8. Chapter 3: Materials and Methods 51

9. Chapter 4: Results 65

10. Chapter 5: Discussion 86

11. Conclusions 106

12. Future prospects 108

13. References 109

14. Appendix

i

LIST OF ABBREVIATIONS

μg/ml Microgram per milliliter

API Analytical profile index

ATCC American type culture collection

bp Base pair

CFU Colony Forming Unit

CLSI Clinical and Laboratory Standard Institute

dNTPs Deoxyribonucleotides

EMB Eosin methylene blue

ESBL Extended-spectrum β-lactamase

GI Gastrointestinal

ICU Intensive Care Uunit

Kb Kilo base pairs

kDA Kilo Dalton

mg/l Milligram/liter

MH Mueller-Hinton

MIC Minimum Inhibitory Concentration

MRSA methicillin-resistant Staphylococcus aureus

NAG N-acetylglucosamine

ii

NAM N-acetylmuramic acid

NCCLS National Committee for Clinical Laboratory Standards

ng Nanogram

OMP outer membrane protein

OPD Out-patient department

PCR Polymerase chain reaction

rpm Revolution per minute

SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

U/ml Unit per Milliliter

UTI urinary tract infections

w/v Weight by volume

μm Micrometer

iii

List of Tables


1 Bacterial DNA concentration (PA15, PA 33, PA 38) 58

2 Oligonucleotide sequence of primers used for the detection

of shiga toxin producing genes.

60

3 Oligonucleotide sequence of primers used for lineage

specific polymorphism Assay

61

4 Oligonucleotide sequence of primers used to detect eae, tir

and hlyA genes

61

5 Oligonucleotide sequence of primers used to detect shiga

toxin bacteriophage insertion sites in stx genes

62

6 Biochemical tests used for the identification of E. coli 66

7 Overall prevalence of diarrheagenic E. coli isolates 72

iv

List of Figures


1 Study flow chart 56

2 Microscopic examination of (a) Diarrheagenic E. coli on

differential media (b) Microscopic image of E. coli (c)

Serotype O157:H7 growth on Sorbitol MacConkey agar

66

3 Serotype O157 positive agglutination test. 67

4 Overall distribution of diarrheagenic E. coli among the study

group

67

5 Prevalence of DEC strains in diarrheal categories 68

6 Overall distribution of diarrheagenic E. coli among different

age categories.

69

7 Gender wise prevalence of diarrheagenic E. coli 70

8 Percentage distribution of diarrheagenic E. coli on the basis

of sample origin

71

9 Annual percentage isolation of diarrheagenic E. coli 73

10 Percent detection of serotype O157:H7 among the isolated

DAEC isolates.

74

11 Seasonal prevalence of DEC pathogroups 75

12 Prevalence of DEC pathogroups in water sources 76

13 Prevalence of ETEC, EPEC and EHEC in water sources 77

14 Prevalence of diarrheagenic E. coli pathotypes in meat

sources

78

15 Prevalence of diarrheagenic E. coli pathotypes in vegetable 79

v

sources

16 Prevalence of ETEC, EPEC and EHEC in vegetable sources 80

17 Antimicrobial resistance patterns of E. coli O157:H7 serotype

isolates.

81

18 Ethidium bromide stained agarose gel showing PCR

fragments for stx1, stx2 and stx2c gene of shiga toxin

producing E. coli, Lane M: 1kb DNA ladder.

82


fragments for eae, hlyA and tir gene of shiga toxin producing

E. coli, Lane M: 1kb DNA ladder.

83


fragments for smfA, rbsB, Arp-icIR, rtcB, z5335 and yhcG of

diarrheagenic E. coli pathotypes, Lane M: 1kb DNA ladder.

83


fragments for stx1, stx2, stx2c, yehV EDL 933,yehV left

junction, wrbA EDL 933, wrbA right junction, sbsB and

argW of diarrheagenic E. coli pathotypes, Lane M: 1kb DNA

ladder

84


fragments for stx1, stx2, stx2c, yehV EDL 933, yehV left

junction, wrbA EDL 933, wrbA right junction, sbsB and

argW of diarrheagenic E. coli pathotypes, Lane M: 1kb DNA

ladder

85

vi

ACKNOWLEDGEMENTS

All praises be to Allah, the most beneficent, the most merciful. His prophet Muhammad

(P.B.U.H), the most perfect of human beings ever born, is the source of guidance and

knowledge for humanity, forever.

I wish to initiate this acknowledgement with the deep indebtedness to Dr. Fariha Hasan,

Chairperson, Associate professor, Department of Microbiology, Quaid-i-Azam

University, Islamabad, Pakistan, who extended full support in planning and execution of

this work, and thesis writing. I appreciate her vast knowledge of microbiology,

understanding of life, unprecedented laboratory and writing skills, and uncompromising

quest for excellence that enabled me to successfully complete this huge task. She was

always there to help me and suggest appropriate remedies, whenever I needed. I would

like to thank her for all the support and motivation she provided during my PhD studies.

The affectionate guidance and whole-hearted cooperation of Prof. Dr. Safia Ahmed, the

Ex-Chairperson, Department of Microbiology, Quaid-i-Azam University, Islamabad, and

sustained academic support of Dr. Aamer Ali Shah, did work wonders in producing and

reforming my research. I must appreciate and MRL staff who were always forth coming

and helpful in rendering any help sought after.

Thanks are also due to Prof. Dr. Abdul Hameed, Ex-Chairman and Dean, Faculty of

Biological Sciences, for his interest in my work and providing the necessary research

facilities. Many thanks are due to members of Pathology Department, Khalifa Gul

Nawaz Hospital, Bannu, especially Dr. Shah Jehan, who provided samples and trained

me in laborious yet very interesting task of data collection and processing.

I would also like to thank Prof. Mark Eppinger, Department of Biology, University of

Texas, San Antonio, USA, for inviting me on a six-month research visit in his lab. This

work would not have seen the light of the day without his guidance, help and mentoring.

vii

My lab fellows at Fatemah Sanjar; Whitney Holhoizer and Yuen Rachel made my stay in

San Antonio, a memorable experience of my life.

I am grateful to Dr. Rahmat Ali Khan for his encouragement, valuable suggestions and

advices at each stage of research work and write up of this thesis.

The co-workers and M. Phil students at MRL, their cheerful presence made my working

very interesting and their intriguing questions kept me thoughtful during our discussions.

My lab fellows and friends at MRL, Fazal, Zia, Sami, Saadia Andleeb, Bashir, Naima,

Maryam, Pir Bux, Zulfiqar, Nida, Farah, Iffat, Sadaf (without prejudice to those whose

names are not annotated) were my real strength in giving me work support, sustained

environment, unbiased positive criticism and all available help. Their moral and material

support eased my resolve to work with dedication and tirelessness.

I would be failing my duty if I do not acknowledge the moral, material and spiritual

support of my loving parents, my brothers, my sisters, and all the other family members

who bore with me during testing times.

Last but not the least, my loving mother, a relaxation and harmony, was source of

inspiration for me. She filled my life with thrill, happiness and joy, and is the most

beautiful gift of my life.

Mir Sadiq Shah

viii

Abstract / Summery

Diarrheagenic Escherichia coli (DEC) pathotypes are ranked third among the list of

infectious agents, due to high morbidity/mortality rate in developed and developing

countries. DEC transmission is linked to the ingestion of contaminated food and water.

Fecal wastes from domestic animals, wildlife and humans are added to the water bodies

via leakage or surface runoff. Humans also contaminate water bodies by adding poorly

treated or untreated sewage effluents, spilling of septic tanks etc. Due to poorly

developed sewage system monsoons rains in Pakistan resulted in heavy floods across the

country affecting 21 million people in 2010. During the said calamity resulting in 5.3

million medical consultations, where 708, 891 13% were made only for acute diarrhea.

Irrigation of agricultural farms with untreated water results in contamination of food

products during harvesting, processing and handling. Foodborne diarrheal outbreaks

were reported due to consumption of green leafy vegetables, drinking water and meat

sources. Complications of Escherichia coli (E. coli) pathotypes infections are ranging

from mild to severe life threatening diarrhea, hemorrhagic colitis and hemolytic uremic

syndrome. This study was conducted to assess the prevalence and molecular

characterization of Diarrheagenic E. coli in Southern Khyber Pakhtunkhawa, Pakistan.

Diarrheagenic E. coli pathotypes were isolated from diarrheal stool specimens,

vegetables, meat and water sources collected for present study (2010-2012). DEC strains

were isolated using (1) MacConkey agar, (2) Eosin Methylene blue agar (EMB), (3)

Sorbitol MacConkey agar and (4) Cefixime Tellurite Sorbitol MacConkey Agar.

Furthermore, E. coil strains were identified through biochemical tests. Colorless, non-

sorbitol fermenting E. coli strains on Sorbitol MacConkey agar were confirmed as E. coli

O157:H7 using DrySpot E. coli O157:H7 agglutination Kit (Oxoid, UK). Diarrheagenic

E. coli pathotypes were also tested for antibiotic resistance using Kirby-Bauer disc

ix

diffusion method. Characteristics virulence factors DEC pathotypes i.e. (1) stx1, (2) stx2,

(3) stx2c, (4) eae, (5) tir, (6) hlyA, (7) est (8) elt and (9) bfpA were detected using

Multiplex PCR. Amplification conditions comprised of 94°C for 6 min, followed by 35

cycles of 94°C for 50 s, 57°C for 40 s and 72°C for 50 s, and finally 72°C for 3 min.

Amplicons were analyzed by electrophoresis on agarose 1.5% w⁄v gels using standard

conditions, followed by staining with ethidium bromide.

(1) Water, (2) Meat and (3) Vegetable samples collected during the present study were

processed for the isolation and identification of diarrheagenic E. coli pathotypes using

differential media, as described above. Multiplex PCR was used the detection of

virulence genes i.e. stx1, stx2, stx2c, eae, tir, hlyA, bfpA, heat stable toxin (ST) and heat

labile toxin (LT). Amplification conditions comprised of 94°C for 6 min, followed by 35

cycles of 94°C for 50 s, 57°C for 40 s and 72°C for 50 s, and finally 72°C for 3 min.

Amplicons were analyzed by electrophoresis on agarose 1.5% w⁄ v gels using standard

conditions, followed by staining with ethidium bromide.

Prevalence of diarrheagenic E. coli was found as high as 57% (515/900) during the

present study. Based on stool physiology, 54.4% E. coli strains were isolated from

watery diarrheal samples, compared to 37.6% from mucoid stool and 8% from bloody

diarrheal stool specimens. Of group I, 155 (30%) isolates were confirmed as E. coli

strains. Similarly, number and percentage isolation of DEC strains from each respective

age group is as follows: Group II 137 (26.6%), Group III 59 (11.4%), Group IV 47

(9.2%), Group V 44 (8.6%), Group VI 42 (8.2%), and Group VII 31 (6%). However,

none of the age groups achieved statistical significance. E. coli strains were obtained

from 40% male patients compared to 60% strains from female patients. The frequency of

E. coli strains recovered different units of hospitals were as; 31.8%, 52%, 3.15% and

12.94% from medical ward, Pediatrics ward, OPD and ICU, respectively. From outdoor

x

patients 44.5% samples were collected from refugee camps survey. E. coli strains from

age Group I, was 14.1% where identified as E. coli O157:H7, followed by Group II:

12.4%, Group III: 13.5%, Group IV: 10.6%, Group V: 9%, Group VI: 14.2% and Group

VII: 6.4%. During the present study, 11.8% (61/515) strains were isolated during winter

season, 21.3% (110/515) during spring season, 47% (242/515) during summer season

and 19.8% during autumn season.

Samples from pond water were contaminated with 28% (14/50) E. coli strains, where

14% (2/14) were identified as serotype O157:H7. Tap water was found to be free of

serotype O157:H7 contamination. Sewage water samples were contaminated with 62%

E. coli strain, where 16.13% (5/31) were identified as serotype O157:H7. Similarly, 38%

(19/50) irrigation water samples were contaminated with E. coli pathotypes, where

13.8% (3/19) were identified as serotype O157:H7. DEC strains 64 isolated from water

sources were randomly selected for pathogroup specific molecular characterization.

Enterotoxigenic E. coli were identified as 56.25% (36/64) of pathogroups including a

combination of heat stable toxin (ST) and heat labile toxin (LT) gene. Enteropathogenic

E. coli were identified as 28% (18/64) comprising of 72% (13/18) typical

enteropathogenic E. coli and 28% (6/18) atypical E. coli. Only 15.6% (10/64) were

identified as E. coli O157:H7 serotype carrying stx1 and stx2 genes.

Beef samples were contaminated with 58% of (29/50) E. coli strains and 20.9% (6/29) of

these were identified as serotype E. coli O157:H7. Chicken meat samples were

contaminated with 16% (8/50) E. coli strains and none of them was identified as serotype

E. coli O157:H7. Sheep meat samples were contaminated with 21.5% (3/14) serotype E.

coli O157:H7. Goat meat samples were contaminated with 34 % (17/50) E. coli

pathotypes, where only one among 17 (5.9%) was confirmed as serotype E. coli

O157:H7.

xi

(1) Mixed salad samples were contaminated with 54% of (27/50) E. coli strains, and

14.8% (4/27) of them were identified as E. coli O157:H7. (2) Cucumber samples were

contaminated with 46% (23/50) E. coli strains and 30.4% (7/23) were confirmed as E.

coli O157:H7. (3) Spinach samples were contaminated with 28% (14/50) E. coli strains

and 28.57% (4/14) were identified as E. coli O157:H7. (4) Lettuce samples were

contaminated with 40% (20/50) E. coli strains and 15% (3/20) were identified as E. coli

O157:H7. STEC isolates (100%) were positive for the presence of stx1, stx2. tir, hly and

eae genes.

Among Shiga toxin, Bacteriophage Insertion (SBI) 100% stx1 bacteriophage was found

to be inserted in yehV gene (both right and left side insertion). stx2 was completely

invaded by the bacteriophages at wrbA site, and similar invasions were observed at sbsB

occupied by stx2C bacteriophage.

Out of 300 DEC isolates, randomly selected for antibiogram development, included 150

(50%) E. coli from outpatients, 75 (25%) from medical ward and 75 (25%) from

pediatrics ward. 94% isolates were found sensitive to imipenem, cefuroxime was the

second most effective antibiotic (55%). The maximum resistance (92%) was observed

against tetracycline, followed by ampicillin 83% and ciprofloxacin 81%. In case of β-

lactam antibiotics, high resistance (78%) was observed against amoxicillin/clavulanic

acid.

Sewage water and industrial effluents needs prior treatment before entering into to the

environment. Drinking water needs to be boiled before intake. Firm adherence to the

prescribed drugs can decrease trends in antibiotics resistance.

Oral rehydration therapy is strongly recommended to minimize extensive dehydration

leading to kidney failure. It is further, recommended that the use of antibiotics in food,

animals should follow prudent guidelines, to minimize spread of resistant bacteria.

xii

Keywords: Escherichia coli (E. coli), Diarrheagenic E. coli (DEC),

Enterohaemorrheagic E. coli (EHEC), Diarrhaegenic E. coli (DEC), Shiga Toxin

producing gene (stx)

Introduction

1

Introduction

Bacteria live in association with other organisms by an agreement and sometime may

cause infections by entering into humans (Levy, 1997). In the history of humankind,

infectious diseases are ranked higher in term of mortality and morbidity. Antibiotics as a

therapeutic agent in the industrialized world, not only shorten the disease period but also

to lower down diseases mortality rate (Yoshikawa, 2002). Antibiotics are regarded as the

most important discovery in the “drugs” development history in the hope for combating

of infectious diseases. On the other hand, silent but sequential changes in bacterial

genome give them a better chance to survive (Levy, 1997). Mutational changes enable

bacteria to resist against routinely prescribed antibiotics leading to an emerging issue of

antibiotic resistance in the last two decades of 20th

century. The inappropriate use of

drugs and self-medication may further encourage the emerging problem of multi-drug

resistant pathogenic (Barbosa and Levy, 2000).

Diarrhea

Diarrheal infections may occur due to alteration of the gastrointestinal tract (GIT)

function by infectious agents or due to metabolic disorders (Guerrant et al., 2005).

Diarrheal infection is defined as “Periodic discharge of unsteady, watery stool, three or

more times in a day. Form and steadiness of the stool is more reliable to define the type

of diarrhea than the number of defecation.

Diarrheal consequences are divided into three categories:

1. Acute watery 2. Persistent diarrhea 3. Bloody diarrhea

In medical terminology acute and persistent diarrhea are not distinct from each other due

to their symptoms, discharge period and morphology of stool. Acute diarrhea is the most

common form, which may resolve within a couple to seven days. Among diarrheal

Introduction

2

infections, acute diarrhea contributes 80% of childhood diarrhea, with rising mortality

rate of about 50% (Wirth et al., 2006).

Persistent diarrhea is less common form, which could extend beyond two to four weeks.

An estimated disease burden of persistent diarrhea is about 10% of all diarrheal episodes

also showed 35% of mortality rate in the developing countries. About 50% of diarrheal

mortality has observed only in South Asia (Khan et al., 1993).

Bloody diarrhea contains visible or microscopic blood cells in the stool, due to evasion

of local mucosal epithelial cells and intestinal hemorrhage (Keusch et al., 2006). A

distinct form of bloody diarrhea is called “dysentery syndrome” characterized by the

presence of blood in small volume of stool, symptomized with abdominal cramps, where

patient feel extreme pressure during fecal discharge. Fifteen percent of deaths are

attributed to dysentery in 10% of all cases (WHO, 2005).

During diarrheal onset, physiological changes may take place in the intestinal tract and

the intestinal physiology is altered by enteric pathogens principally in one of three ways:

1. By changing water and electrolyte fluxes in the upper small intestine, often resulting

in the form of watery diarrhea.

2. Through inflammatory or cytotoxic effect on the intestinal mucosa cells, resulting in

the release of leukocytes in the stool.

3. By microbial invasion through an intact mucosal membrane to the underlying

reticuloendothelial system (Guerrant et al., 2002).

Gastrointestinal tract (GIT) Infections are among the leading causes of mortality among

children and adults. These infections were reported to cause more than 3.2 disease

episodes per year in children/adults in developing countries (Kosek et al., 2003).

Introduction

3

Escherichia coli

Escherichia coli (E. coli) are Gram negative bacterium belongs to the family of

Enterobacteriaceae. E. coli are motile, catalase positive, oxidase negative, ferment

glucose, but do not ferment sorbitol and glucuronidase, reduce nitrate, and indole

positive. E. coli may also cause a series of gastrointestinal tract infections (GIT). E. coli

may cause urinary tract infections (UTIs) complications in the community settings and

hospitals environment (Stamm and Hooton, 1993). Occurrence of characteristics

virulence genes aids in the adaptation of pathogenic E. coli either in attachment or

invasion (Kaper et al., 2004). One of these factors is enterohaemolysine a (eae) relate to

colonization and fitness making the pathogen able to attach and invade host surface

mucosal layer. The second virulence factor is shiga toxin producing genes (stx1, stx2)

which are responsible for the release of shiga like toxins.

E. coli strains are categorized into pathogenic and nonpathogenic strains based on

characteristics virulence factors.

1. Enterotoxigenic E. coli (ETEC), 2. Enteropathogenic E. coli (EPEC),

3. Enteroheamorragic E. coli (EHEC), 4. Enteroaggregative E. coli (EAEC), 5.

Enteroinvasive E. coli (EIEC) 6. Diffusely Adhering E. coli (DAEC) (Rappelli et al.,

2005).

Disease pattern of diarrheagenic E. coli varied due to the difference in virulence genes.

Persistent diarrhea is caused either by EAEC or EPEC, invasive dysentery is caused

EIEC or Shigella, while bloody diarrhea caused by EHEC and STEC. Complex diarrheal

infection is caused by mixed pathogens including bacteria and protozoans. For such type

of infections antimicrobials therapy should not be used. Unfortunately, antibiotics are

commonly prescribed for all type of diarrheal infections. Prescription would be effective

Introduction

4

is Physicians could discriminate which infections are self-resolving and which might be

life threatening.

Enteropathogenic E. coli (EPEC)

Diarrheal infections are escalating in the developing countries with high

morbidity/mortality rate. EPEC form attaching and effacing (A/E) lesions on the host

intestinal epithelial cells responsible for gradual erosion of underlying microvilli and

disruption of host cell actin, forming distinct pedestals on the site of attachment.

Formation of A/E lesions in EPEC is due to presence of eae genes, on LEE (locus of

enterocyte effacement) encrypted on 35 kb PAI (Pathogenicity Island) (McDaniel et al.,

1995). Type III secretion system (T3SS) is activated by the LEE that result in the

transport of bacterial toxins into the cytoplasm of the attached cell. Several effector

proteins are determined by the LEE, while other than LEE encoded (Nle) proteins can

also found as well, although their roles are unknown (Deng et al., 2004).

EPEC binds to the host intestinal enterocytes using bundle-forming pili (bfpA) present on

the EPEC adherence factor (EAF) plasmid (Hyland et al., 2008). Attachment to the

target cell is brought through intimin (bacterial outer-membrane protein) and Tir

(translocated intimin receptor) (Kenny et al., 1997). Tir brought Nck to the site of

attachment (Gruenheid, DeVinney et al., 2001), activating neural wiskott–Aldrich

syndrome protein (N-WASP) along with actin-related protein 2/3 (ARP2/3) complex to

initiate actin assembly and specific pedestal formation (Kalman et al., 1999).

Enterohaemorrheagic E. coli (EHEC)

During the past two decades, EHEC gained considerable importance by registering

numerous foodborne outbreaks. EHEC inhibit distal part of the large intestine in humans

Introduction

5

resulting in bloody diarrhea, sometimes may lead to hemorrhagic colitis (HC) or

hemolytic uremic syndrome (HUS) (Kaper et al., 2004). EHEC cycle is completed

through vegetables, meat sources and water used for irrigation. Enterohaemorrheagic E.

coli O157:H7 isolates harbor a 92 kb pO157 plasmid, containing 100 open reading

frames having several virulence genes. Characteristics virulence genes of shiga toxin-

producing E. coli (STEC) is the bacteriophage-encoded shiga toxin (Stx or

verocytotoxin) (Asadulghani et al., 2009; Nataro and Kaper, 1998). Structurally shiga

toxin is an AB5 type toxin, where B subunit (pentamer) is non-covalently attached to A

subunit. There exists no secretary mechanism for the release of shiga toxin in EHEC

(Toshima et al., 2007). More effective toxin would be released against antibiotic therapy

as in response to the DNA damage. Globotriaosylceramides (gb3s) are another stx

receptors are found on host paneth cells (Toshima et al., 2007). Cattle lack gb3s

receptors in their gastrointestinal mucosa, and thereby remain asymptomatic because of

EHEC inability to colonize in cattle (Pruimboom-Brees, Morgan et al., 2000). The shiga

toxin pentamer (stxB) subunit attached with Gb3 receptor causes membrane

internalization to simplify toxin entry (Romer et al., 2007). The internalized shiga toxin

A subunit (N-glycosidase) is activated by a cleavage event (Kurmanova et al., 2007),

leading to necrosis and cell death (Gobert et al., 2007).

Enterotoxigenic E. coli (ETEC)

Travellers‟ diarrhea is commonly ETEC both in industrialized and under developed

countries. ETEC is fatal in children due to severe dehydration. ETEC may harm

livestock farming, as young piglets are mostly infected by ETEC infections (Nataro and

Kaper, 1998). Colonization factors (CFs) are used by ETEC for the attachment to the

host epithelial cells, which are fimbrial, non-fimbrial, helical or fibrillar in nature. More

Introduction

6

specifically ETEC intimate attachment to the host cell may be due to the outer-

membrane proteins Tia and TibA (Turner et al., 2006). Diarrheal infection due ETEC

pathotypes may occur due to the secretion of heat-stable enterotoxins (STs), and heat-

labile enterotoxin (LT) in gastrointestinal tract. Based on the structure and function Heat-

stable enterotoxins STs are minor molecular toxins categorized in STa or STb (Turner et

al., 2006). Heat labile toxin is like cholera toxin similar AB5 toxin. The B subunit of LT

interacts with the mono-sialoganglioside GM1 on host cells; the toxin is entered through

lipid rafts (Kesty et al., 2004), where it reached to the cytoplasm after passing through

the endoplasmic reticulum.

Enteroinvasive E. coli (EIEC)

It is commonly believed that EIEC and Shigella belongs to the same origin, due to

similar mechanisms of pathogenicity. Virulence is mainly attributed to a 220 kb plasmid,

which a type 3 secretion system used for membrane internalization (Ogawa et al., 2008).

Enteroaggregative E. coli (EIEC)

Traveller‟s diarrhea is mostly caused by EAEC both in developed and as well as

developing countries. EAEC always cause watery diarrhea that could contain small

amount of mucus or blood. EAEC primarily attached to the mucosa of both small and

large intestines, by causing mild colon inflammation. EAEC is differentiated based on its

stacked brick pattern of attachment to HEp-2 cell from other pathovars. Adherence

Aggregative plasmid (pAA) plasmid is involved in the synthesis of the aggregative

adherence fimbriae (AAFs), and attachment of EAEC to the host intestinal cells (Nataro

and Kaper, 1998).

Introduction

7

Diffusely Adherent E. coli (DAEC)

DAEC utilize unique binding pattern on a layer of HeLa and HEp-2 cells. Adherence is

to the host cell is facilitated by a group of related proteins including Dr and F1845 and

Afa adhesions. DAEC pathotypes cause diarrhea in young children and may cause

urinary tract infections (UTIs) in adults carrying either Afa-Dr adhesions. DAEC on

attachment to DAF recruits all of its underlying molecules. DAEC also activates a Ca2+

dependent cell signaling cascade triggering impairment of brush border microvilli

through the dissociation cytoskeleton components (Servin, 2005).

Uropathogenic E. coli (UPEC)

UPEC is involved in 80% of UTI infections, triggering cystitis in the urinary bladder and

onset of pyelonephritis in the kidneys. For causing an infection UPEC travel all the way

down to the urinary tract from GIT, using proteins and amino acids as a source of energy

(Maruvada et al., 2008). The ability of UPEC to reach kidney against the concentration

gradient from urethra to the urinary bladder, where it showed distinct mechanisms for

organ tropism, escaping inborn immunity and dodging tackling by macrophages. This

complex mode of pathogenesis is strictly regulated by a combination of several virulence

factors i.e. multiple pili, secreted toxins (Sat) and vaculating autotransporter toxin (Vat)

(Wiles et al., 2008).

The Evolution of Pathogenic E. coli

E. coli is harmlessly remained in the intestinal tract and helps the host by production of

Vitamin B12. Pathogenic strains evolved by gaining genetic elements through horizontal

gene transfer. Pathogenic strains are highly diverse in their genome composition by the

progressive gaining and removal of genetic elements with the passage of time. Genetic

Introduction

8

alteration might have been took place about 9 million years ago with the attaining of

pathogenicity sequences from other bacteria through Horizontal gene Transfer (HGT)

(e.g. via bacteriophages, transposons and plasmids) which is still evolving. Therefore,

evolution of E. coli is due to the acquisition of novel genetic materials from other

organism like shigella. Based on its universal existence, E. coli needs to further

modifications to grow in such dynamic environmental conditions. To combat these

environmental changes and host immune response E. coli almost relies on the

diversification of genetic element. Acquisition of novel genetic material brings genome

plasticity and provides increased chance of adaptability. These acquired genes are

located on plasmids, enabling bacteria to rapidly share and obtain to produce new

bacterial strains. Emerging new bacterial variants tends to stabilize, acquiring more

genetic diversification that may leads to the development of new strains.

E. coli O157:H7

E. coli O157:H7 is most frequently isolated from patients suffering from bloody

diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (Benjamin and Datta,

1995; Kaper, 1998; Yoon and Hovde, 2008). Other members of EHEC family causing

hemorrhagic colitis are E. coli O26:H11 (Robins-Browne, 1995). 10-80% of the cattle

serve as primary host for E. coli O157:H7, colonizing in the last part of large intestine

(Hussein and Sakuma, 2005; Welinder-Olsson and Kaijser, 2005). All cattle except

young calves remain asymptomatic to the E. coli O157:H7 infection. Presence of E. coli

O157:H7 in ruminants is beneficially highlighted by several studies (Hussein and

Sakuma, 2005). Contrary, human beings are highly vulnerable to E. coli O157:H7

infection, acquiring it from fecally contaminated food or water (Moxley, 2004).

Introduction

9

Evolution of E. coli O157:H7

Enterohaemorrheagic E. coli (EHEC) O157:H7 is thought to have evolved from a

sorbitol fermenting and β-glucuronidase positive O55:H7 ancestors. The genomic loci of

cardinal virulence factors of the EHEC strains indicate that virulence factors are mainly

acquired through mobile genetic elements. Acquired elements are LEEs, lambdoid

phages, insertion sequence elements, and pEHEC plasmids etc.

The mechanisms underlying the evolution of these virulence factors in EHEC strain are

not documented. It is believed that subsequent addition or deletion of genetic material

have profoundly influenced E. coli genome. Comparison of the genomic sequence of

EHEC O157 with the non-pathogenic laboratory strain K-12 genome has revealed the

acquisition of foreign genetic materials has played a vital in the evolution of EHEC

O157 (Hayashi et al., 2001; Perna et al., 2001). Genomic comparison of these strains

showed the conservation of 4.1-Mb sequence, a region like the backbone of the E. coli

genome, while rest of the 1.4-Mb sequence consists of EHEC O157 specific sequences.

Genetic heterogeneity among EHEC O157:H7 strains have been established using a

broad panel of typing methodologies, such as multilocus sequence typing (Rajkhowa et

al., 2010), octamer- and PCR-based genome scanning (Kim et al., 1999), phage typing,

multiple-locus variable number tandem repeat analysis (Keys et al., 2005), microarrays

(Bochner et al., 2001; Zhou et al., 2003), nucleotide polymorphism assays (Syvanen,

2001), pulsed-field gel electrophoresis (PFGE), subtractive hybridization, and whole

genome mapping (Zhou et al., 2004; Latreille et al., 2007; Zhou et al., 2007;Wu et al.,

2009).

Introduction

10

Infectious Dose of E. coli O157:H7

E. coli O157: H7 are isolated from contaminated food and water associated with disease

outbreaks as low as 10–100 CFU/g or CFU/ml of the food or water analyzed. Even this

low number of cells is sufficient to show disease signs and symptoms in the host (Mead

and Griffin 1998).

Pathogenicity of E. coli O157:H7

The pathogenicity of E. coli O157:H7 is attributed to its virulence factors i.e. periplasmic

catalase and Shiga-like toxins (Nataro and Kaper, 1998). Shiga toxins may inhibit

protein synthesis and induce apoptosis by blocking 28S ribosomal RNA subunits of

eukaryotic cells (Reisberg and Rossen, 1981). The periplasmic catalase may aid in the

oxidative protection of the pathogen during host infection (Brunder et al., 1999). The

major disease causing E. coli virulence factors in human infections include shiga toxin

antigen production (Stx1 and Stx2 variety) and tight adherence to host epithelial cells of

the large intestine using secretory proteins encoded in the pathogenicity island called

locus of enterocyte effacement (LEE) (e.g. eae, tir, Type III secretion system) (Perna et

al., 1998; Cornick et al., 2000; Chaisri et al., 2001; Hayashi et al., 2001; Eklund et al.,

2002; de Sablet et al., 2008; Abu-Ali et al., 2010; Chen et al., 2013). The main function

of stx is cleaving of the 28S rRNA such that it leads to inhibition of binding between

tRNA and 60S rRNA and disruption of peptide elongation during protein synthesis

(Hofmann, 1993, Rahal et al., 2012). The gastrointestinal infection caused by E. coli

O157:H7 may lead to HUS and HC in humans due to the absorption of the stx through

the gut and subsequent glomerular vascular damage (Poirier et al., 2008; Park et al.,

2013). The pro-inflammatory response of the host leads to micro-vascular thrombosis

and damage of the red blood cells. The progression of these symptoms without treatment

Introduction

11

especially in the elderly and infants results in hemolytic anemia, renal function

disruption, and an ultimate death. The LEE is a 35-kb cluster of genes that encode

virulence associated factors including the Type III Secretion System (T3SS), eae, and tir

protein (Perna et al., 1998; Hayashi et al., 2001; Coburn et al., 2007). The T3SS is

essential for establishing of EHEC infection by causing attaching and effacing (A/E)

lesion in host epithelial cells (Jerse et al., 1991; Perna et al., 1998). The LEE aids in the

attachment of E. coli to the host epithelial cells and induction of host signal transduction

pathways (Perna et al., 1998).

Foodborne Outbreaks of E. coli O157:H7

Since 1982, numerous foodborne outbreaks from USA, Europe, Asia and Africa have

been reported by this bacterium (Ihekweazu et al., 2006; Mashood and Simeen, 2006). In

1996, separate Foodborne E. coli O157:H7 outbreaks were reported from British

Columbia, California, Colorado and Washington (Cody et al., 1999a) followed by

another E. coli O157:H7 outbreak in 2002 owing to the consumption of ground beef

(Vogt and Dippold, 2005).

Sporadic foodborne outbreaks of E. coli O157:H7 were reported in UK (Parry & Salmon,

1998). Similarly, another outbreak was reported in 2002, due to the ingestion of

cucumber and hot-dog pastry that the students fed on during the trip (Duffell et al.,

2003). In August 2004, E. coli O157 infections were identified in children in Southwest

England. The outbreak was suspected on a contaminated freshwater stream flowing

across a seaside beach (Ihekweazu et al., 2006). Contaminated radish sprouts borne E.

coli O157:H7 outbreak has been reported in Sweden (Soderstrom et al., 2005) along with

radish sprout salad borne outbreak of E. coli O157:H7 in Japan (Watanabe et al., 1999).

Introduction

12

Epidemiological data on E. coli O157: H7 in developing countries is not well

documented, as surveillance for this pathogen is not done routinely.

Seasonal Prevalence of Diarrheagenic E. coli

The importance of seasonal variation on community-associated infections is reported for

influenza infection. Higher seasonal infection trends have been observed in opportunistic

pathogens like E. coli. One can better devise public health policy by estimating the

environmental factors i.e. warm season and humidity that aids in the survival and

optimum growth of these pathogens. Rota viral infection became double the winter

season in Mexico. Similarly, rates of pediatric diarrhea in the US also vary seasonally,

with viral diarrhea predominating in winter season, and bacterial infections are seen

during springtime. Diarrheal infections are most commonly observed during summer

than during winter months.

Transmission of E. coli

Water

The recent flood has affected about 78 districts in Pakistan, affecting 21 million people.

This catastrophe was the biggest after 2005 earthquake, which left people of affected

areas in a horrible situation. According to (WHO) World Health Organization 2010

report, flooding has affected 21 million people. Flooding cause 5.3 million medical

consultations, where 708, 891 (13%) were made for acute diarrhea. During this natural

disaster, fecal contaminants were added to the water bodies i.e. untreated or poorly

treated sewage water, direct leakage of septic tanks and spillage from sanitary passages

(Cody et al., 1999). Waterborne diarrheal outbreaks due to E. coli pathogroups may

occur due to the fecal contamination (Schets et al., 2005). Recreational water sources can

Introduction

13

be also contaminated with these pathogroups (Verma et al., 2007). Activities of these E.

coli pathotypes resulting in an outbreak has been found throughout the UK, USA,

Canada, Japan, Sweden to name but a few (Woodward et al., 2002; Cagney and

Browning, 2004; Sartz et al., 2008; Uhlich et al., 2008; Chen et al., 2013).

Meat and its Products

Occurrence of E. coli O157:H7 have been reported from cattle, their carcasses, hides and

feces (Brichta-Harhay et al., 2007). 15.7% prevalence rate of E. coli O157:H7 in cattle

have been reported from UK (Chapman et al., 1997). Prevalence of E. coli O157:H7 is

rarely documented in Pakistan. Studies on E. coli O157:H7 in bovine and their meat

products showed 8-15.7% in cows (Wells et al., 1991; Chapman et al., 1997) and 1.8%

in cattle herd (Hancock et al., 1997). E. coli O157:H7 infection rate in animals varied

from 0-60% (Blanco et al., 1996). However, prevalence of E. coli O157 in cattle and

their carcasses was much higher than any other (Elder et al., 2000). It is evident that the

prevalence rate could be lower down by following standard sanitary procedures (Elder et

al., 2000). There is seasonal variation in E. coli O157:H7 shedding in cattle. Fecal

shedding of E. coli O157:H7 varied from low to high depending on the season

(Edrington et al., 2006). High proportion of E. coli O157:H7 is reported during the

summer season in cattle (Elder et al., 2000).

Undercooked Ground Meat and its Products

Foodborne E. coli O157:H7 outbreaks have also been reported due to the consumption of

undercooked ground (Riley et al., 1983; Kassenborg et al., 2004). Epidemiological data

shows that the prime risk foods of bovine origin are undercooked hamburgers (Reilly,

Introduction

14

1998). Such contaminated hamburgers or ground beef tend to possess a characteristic

pink color at the middle (Riley et al., 1983).

Milk and its Products

The contamination of milk by E. coli O157:H7 is often suspected to occur during the

milking process. Contamination of milk occurs during collection or processing. Dairy

products have been involved in E. coli O157:H7 disease outbreaks (Karmali et al., 1988).

E. coli O157:H7 were isolated from cheese sandwiches (Kassenborg et al., 2004). This

has been possible most probably because the organism is acid tolerant and can grow

under low acidic condition (Conner and Kotrola, 1995). Different food items that act as a

vehicles for E. coli O157:H7 outbreak are like processed fermented foods such as

yoghurt, cheese and sausage, have been involved in food-borne outbreaks caused by E.

coli O157:H7 (Gansheroff and O'Brien, 2000).

Fruits, Vegetables and their Products

Plants provide habitats for many species of microorganisms such as bacteria, yeasts and

filamentous fungi (Lindow and Brandl, 2003). According to Zhang (Zhang et al., 2010),

bacterial populations on spinach and rape phyllosphere were larger compared to celery,

broccoli, and cauliflower. Human pathogens, principally, E. coli O157:H7 and

Salmonella are transient residents of plants. To survive and grow in the plant

environment, human pathogens have to compete with indigenous members of plant

microbial communities (Brandl, 2006) for nutrition, energy and colonization on the host.

The presence of human pathogens on edible plants can be a source of infection. Many

foodborne outbreaks occur mainly due to the consumption of E. coli and salmonella

contaminated fruits and vegetables. In the US, 21% of E. coli O157:H7 outbreaks occur

Introduction

15

due to the consumption of vegetable products from 1991 to 2002 (Rangel et al., 2005),

and from 1996 to 2007, 33 outbreaks occur with salmonella contaminated fruits and

vegetables (Callaway et al., 2003).

Control of E. coli O157:H7 Food Borne Outbreaks

Foodborne disease outbreaks are attributed to the food items obtained from unsafe

sources, contaminated raw food items, poor food storage, and improper personal hygiene

status during food preparation. Contamination may also occur due to the inadequate

cleanliness of kitchen, equipment and utensils. Undercooking may also lead to the food

contamination, cooling and reheating of raw food and prolonged time lapse between

cooking and eating of the foods. All these factors are responsible for numerous outbreaks

in both developed and developing countries (Kassenborg et al., 2004; Rangel et al.,

2005).

On Farm Control

Control measures must be adopted at the dairy farms and nursery homes, since it is

evident that cattle acts as primary host for E. coli O157:H7 (Lejeune et al., 2004). Diet

management has also been reported to be helpful in controlling the prevalence of E. coli

O157:H7 in cattle.

Use of Antibiotics

The use of antibiotics in the farm animals as a growth promoter and yield enhancement

are under strong criticism, which is expected to continue in the near future (Callaway et

al., 2003; Panos et al., 2006). Extensive ad misuse of antibiotics as a therapeutic agent

and in agriculture has led to the wide spread transmission of antibiotic resistance genes

Introduction

16

in the target pathogens through horizontal gene transfer (HGT) (Van den Bogaard and

Stobberingh 1999).

Vaccination

Vaccination can play a crucial role in the prevention of infection and gave one a better

chance live. Immunization can greatly reduce the disease burden of E. coli, by

eliminating pathogen shedding and colonization. Low shedding of E. coli O157:H7 by

vaccinated calves have been reported in Canada (Finlay, 2010).

Antimicrobial Resistance in Microorganisms

The mechanisms of antimicrobial resistance are described by using four categories:

„Bypass‟ is based on the structural nature of microorganisms‟ outer membrane

which is considered to be a barrier for drug‟s entry into the cell. The outer cell

wall makes these bacteria resistant to many antimicrobials (Okazaki et al., 1994).

Enzymatic inactivation or modification of the antimicrobial. For example, by the

production of the β-lactamase enzymes in E. coli (Jacoby, 1994), which destroys

the β-lactam-ring‟s chemical structure of the penicillin; or, aminoglycoside-

modifying enzymes in Staphylococcus aureus (Kawamura et al., 2001).

The organism has developed antimicrobial resistance through structural changes

of the drug target sites for example, mutations in the genes lead to a structure of

changed drug target sites that inhibit drugs from binding to these target sites

(Webber and Piddock, 2001).

The efflux pump is a self-defense mechanism of a microorganism throwing out of

the drug entering through cell membrane, before it can contact the action site and exert

its effect (Tenover, 2006).

Introduction

17

The efflux system is considered as a pump because the ejection process requires energy.

This mechanism exists in many situations of natural resistance of specific organisms

and in many kinds of antimicrobials such as tetracycline flouroquinolones (Bal et al.,

2010).

Antimicrobial resistance can be classified as either natural resistance or acquired

resistance (Todar, 2002). The natural resistance refers to an organism inherent ability for

resisting an antimicrobial. An example for this is the inherent resistance of E. coli to

penicillin G because there is no reaction site of penicillin G in its structure (Kim et al.,

2005). The acquired resistance refers to a qualitative alteration of the genetic material of

the organism as the result of microbes changing in some ways to eliminate the

effectiveness of drugs through mutations. A mutation in the gyrA gene of E. coli leads to

the result that ciprofloxacin cannot bound an essential bacterial enzyme required for the

DNA replication (Rodriguez-Villalobos et al., 2005).

Introduction

18

AIMS AND OBJECTIVES

Isolation and identification of E. coil pathotypes from clinical stool specimens.

Isolation of Diarrheagenic E. coli from environmental and food samples for

comparison/route of transmission

Primary pathogroups specific screening of diarrheagenic E. coli.

Determination of antibiotic susceptibility pattern of diarrheagenic E. coli

pathotypes.

Frequency distribution and relationship of diarrheagenic E. coli with Age,

Gender, sample origin and sample source.

Serotyping of E. coli O157:H7.

Molecular characterization of diarrheagenic E. coli pathotypes (eae, tir, hlyA, LT,

ST and bfpA) along with shiga toxin producing genes (stx1, stx2, and stx2

variants) in E. coli O157:H7 serotype using multiplex PCR.

Determination of bacteriophage insertion sites in E. coli O157:H7 serotype.

Chapter 2 Review of Literature

19

Review of Literature

The recent flood has affected about 78 districts in Pakistan, affecting 21 million people.


areas in a horrible situation. According to World Health Organization (WHO) 2010


consultations, where 708, 891 (13%) were made for acute diarrhea. This calamity left

long lasting impressions on the health of millions of affected people. During this natural

disaster, fecal contaminants were added to the water bodies like untreated or poorly

treated sewage water, direct leakage of septic tanks and spillage from sanitary passages.

Diarrhea

Diarrheal consequences are often accompanied by a series of symptoms including,

passage of unusually less controlled, formless and watery stools, at least three times

(episode) in a day. A progressive change in the structural uniformity and physical

appearances of the stools are clinically as important as the number of stools passage.

Diarrhea is actually a symptomatic reflection of GIT complications (also termed as

infectious gastroenteritis), by infectious agents and non-infectious conditions (Guerrant

et al., 2002).

In general diarrheal infection is categorized into three categories; acute watery,

persistent, and bloody diarrhea. Former two forms of diarrhea i.e. acute and persistent,

are not distinct from each other based on number of stools discharge and physical

appearance, but generally represent two ends of a continuum. Periodic episodes of acute

diarrhea resolve within the defined disease length (seven days period) but sometimes it

could prolong beyond three to four weeks (McAuliffe et al., 1986).

According to world health, organization (WHO) persistent diarrhea is defined as

“diarrhea lasting 14 days or slightly longer. Even though 14 days limit is arbitrary, it is


20

usually supported by an increased mortality rate in children under the age of 5 years with

a persistent diarrhea of longer duration (Alam and Ashraf, 2003). Bloody diarrhea is

defined as “diarrhea where stools containing visible or microscopic blood, due to local

evasion of mucosal lining or may be due to intestinal hemorrhage (Keusch et al., 2006).

Another distinct form of bloody diarrhea “dysentery syndrome” characterized by small-

volume, bloody stools, accompanied by abdominal cramps, and intestinal tenesmus,

exerting a severe pain during stool discharge.

Generally speaking, of these defined categories, acute watery diarrhea is most common

type of childhood diarrhea, windup with 50% mortality rate, of all 80% registered cases

both in developed and under developed countries. Periodically persistent diarrhea is

experienced in 10% of diarrheal episodes, represented with excessively higher mortality

rate. Altogether, persistent diarrhea contributes about 35% of diarrheal mortality, this

ratio is found somewhat about 50% in South Asia as shown in several studies (Bhan et

al., 1989). Dysentery is seen in 10% of all cases of diarrhea and causes 15% of the

deaths (Khan et al., 1993).

Mechanism of Diarrheal Infection

Diarrhea is thought to be due to the change in the intestinal physiology and function. The

intestinal physiology is altered by enteric pathogens, principally in one of the three ways:

1) Changing water and electrolyte concentration/fluxes in the upper small intestine

resulting in watery diarrhea.

2) Through inflammatory or cytotoxic destruction of the intestinal epithelial mucosa

expressed by the presence of fecal leukocytes and dysentery.

3) By invasion via an intact mucosa to the inner reticuloendothelial system,

progressing to enteric fever starting with mild diarrhea (Bushen et al., 2004).


21

Infectious Diarrhea (ID)

Infectious diarrhea (ID) is a major cause of morbidity and mortality both in developed

and under developed world. Infectious diarrhea could be due bacteria, viruses,

protozoans etc. These agents get an easy access to the GIT through contaminated food

and water. This problem is even worse in developing or low-income countries like

Pakistan, where there is no or underdeveloped sewerage system. Contaminants get an

easy an uninterrupted access to the drinking water and other food resources, thereby

causing varieties of infections like; mild to severe bloody diarrhea. Developing countries

is considered to be the nourishing region where it is estimated to be the major public

health concern with an estimated annual prevalence rate of 1.4 billion episodes among

children less than 5 years of age (Parashar et al., 2003). Of these 123.6 million episodes

of ID are severe enough that seeks medical consultation or outpatient medical care, while

9 million episodes need ultimate hospitalization. Diarrheal infections are even more

severe in young and immune-compromised individuals. Average number of diarrheal

episodes experienced by children less than 5 years of age is 3.2 with highest rate of 4.8

episodes occurring in the first year of life. With the growing age, immune system gets

strengthened, thereby, rate of diarrheal episode decline progressively to 1.4 episodes per

year at 5 years of age. Infantile diarrhea is highly fatal during 1st year of life, with a

mortality rate of (8.5 children per 1000/year) (Kosek et al., 2003). Furthermore, 12600

deaths are attributed to diarrheal infections in children less than 5, each day in Asia,

Africa and Latin America (Nguyen et al., 2006). Kosek et al., 2003 conclude that during

(1990 to 2000) 21% (2.5 million) infantile deaths were attributed to diarrheal infections

in developing countries.

Improved strategies are needed to be implemented in sewerage system, balanced diet,

and oral rehydration therapy among others. A combination of all these preventive


22

measures has lowered down the acquired disease burden of infectious diarrhea (ID) from

4.6 million to 1.5 to 2.5 million since 1982. According to WHO diarrhea is reported,

second most common cause of infantile deaths (O'Ryan et al., 2010). Infectious diarrhea

is usually caused by a number of microorganisms including bacteria, viruses and

parasites (Kosek et al., 2003; Al-Gallas et al., 2007). Studies have suggested that

diarrheagenic E. coli (DEC) pathotypes are the most common bacterial pathogens

associated with infectious diarrhea (ID) in developing countries. Geographic distribution

play an important role in the frequencies of these pathogens depending the

socioeconomic/sanitary conditions and 30-40% of children under 5 years of age

experience diarrhea (Albert et al., 1999; O'Ryan et al., 2010), and about 15-30% have

required hospital care (Nataro and Kaper, 1998; O'Ryan et al., 2005).

In Pakistan, ID is recognized as the common health problem preceded only by the

respiratory diseases, whereas 1/3 of child deaths are attributed to diarrhea. Annually

230,000 Pakistani children (under 5) died because of diarrhea. E. coli have been

implicated as an agent of diarrheal disease since the 1920s (Nguyen et al., 2005).

Symptoms of Bacterial Diarrhea

Watery Diarrhea

Bacterial and non-bacterial pathogens causes clinically nonspecific acute watery

diarrhea. Acute watery diarrhea is defined as “discharge of indistinct 3 or more stools

times in 24 hr period with or without symptoms”. Acute watery diarrhea sometimes also

caused by Salmonella and Campylobacter (Voetsch et al., 2004). Potential infectious

agents are Enterotoxigenic E. coli, Enteroaggregative E. coli, Enteroinvasive E. coli, non

Choleraic Vibrios, and Noro virus.


23

Dysentery

Dysentery or bacterial colitis is characterized by the excretion of bloody stools. Bloody

diarrhea is caused by either Shigella, Campylobacter, non-typhoid Salmonella, and Shiga

like toxin-producing E. coli. Other organisms like Aeromonas species, non-choleric

vibrios, and Yersinia enterocolitica may cause bacterial colitis (Talan et al., 2001).

STEC diarrheal infection onset with watery diarrhea that may leads bloody in several

days in most patients. Symptoms of STEC infection varied from severe abdominal pain

to cramps and discharge of more than five unsteady stools in a day (Tarr, 2009). STEC

induced diarrheal disease may progressively leads to kidney failure in young children.

Shiga toxin discharged in GIT may reach to the renal endothelium via bloodstream in

hemolytic uremic syndrome. Hemolytic uremic syndrome require kidney dialysis which

has mortality rate of 3 to 5% in young children (Kendrick et al., 2007)

Traveler’s Diarrhea

Traveler‟s diarrhea (TD) is defined as “an infection that may occurs due to the

consumption of contaminated food or water in the under developed tropical and

semitropical areas”. TD (80%) caused by the bacterial enteropathogens (Shah et al.,

2009). TD is mostly observed due to the Enterotoxigenic, Enteroaggregative and

Diffusely Adherent E. coli in Latin America, Africa, and South Asia (the Indian

subcontinent). Traveler‟s diarrhea is also caused by other bacterial strains like Shigella,

Salmonella, Campylobacter and Plesiomonas. Pathogenic E. coli are often involved in

diarrheal infections in South Asia and Southeast Asia, compared to other bacterial strains

(Campylobacter, Shigella, and Salmonella).


24

Nosocomial Diarrhea

Diarrheal infection normally occurs in community setting or hospitals, where patients are

treated with drugs and feedings, thereby exposed to Clostridium difficile spores. C.

difficile is involved in antibiotic-associated and hospital acquired diarrheal infection,

toxic dilatation of the colon or otherwise unexplained leukocytosis, or both. Such type of

infection is symptomized with the excretion of watery stools that may progress to bloody

stools.

E. coli as Diarrheal agent- A Historical Perspective

The capability of E. coli strains to cause diarrhea was reported as early as 1887 (Clarke

et al., 2002). However, only after Bray reported the isolation of E. coli from cases of

summer diarrhea in 1945, more widespread interest in this organism as enteric pathogen

evoked. Over the next few decades, E. coli strains of certain serotypes (O111, O55 and

O127) were frequently diagnosed as cause of childhood diarrhea in industrialized

countries (Levine and Edelman, 1984).

The term enteropathogenic E. coli (EPEC) was first introduced 1995 to describe E. coli

strains that were epidemiologically implicated in infant diarrhea (Neter, Westphal,

Luderitz, Gino, & Gorzynski, 1955). During the same period mortality rates >50% were

reported in diarrheal outbreaks among children attributed to EPEC. Since 1960, both the

incidence and lethality of EPEC declined in industrialized countries by the introduction

and intensive use of oral rehydration therapy (Nataro and Kaper, 1998). In Norway,

during this period EPEC was most frequently associated with mild symptoms, and was

diagnosed in hospitalized children (Kvittingen, 1966).

The pathogenic nature of the E. coli strains most frequently diagnosed in children with

acute watery or persistent diarrhea was confirmed in several volunteer studies during the


25

first half of the 1950s (Levine et al., 1985). Furthermore, these strains were usually

isolated in pure culture in children with diarrhea, compared to only in small numbers in

healthy children. E. coli strains, obtained from infantile diarrhea were also positive in

developing serologic response (Nataro, 2006). Other EPEC serogroups like O-serogroups

and O: H serotypes were also responsible for infant diarrhea, and thus were accordingly

classified as classical EPEC serogroups and serotypes based on the presence of

characteristics virulence factors (Levine and Edelman, 1984; DuPont, 1995). However,

the pathogenicity of EPEC strains without any of these virulence factors was definitely

verified in human volunteer studies in (Levine et al., 1978).

Other strains of E. coli carrying heat-labile and/or heat stable enterotoxins were

classified as Enterotoxigenic E. coli (ETEC), where some invasive strains were named

Enteroinvasive E. coli (EIEC) based on their invasive nature. The E. coli strains were

observed to share many virulence strategies to cause variety of infections through

horizontal gene transfer (HGT). Binding to the host cells is required for all E. coli

pathotypes except EIEC. This characteristic is due to the presence of eae gene, and is

brought about by long appendages called fimbriae or pili. After binding to the cell

membrane E. coli must destabilize host cell by disrupting its functions, using

enterotoxins. Forceful disruption of host cell signaling pathways is another strategy used

for the invasion/dominating of host cells immune system and competent colonization.

Coordination of these combined strategies ultimately leads to disease. Each class of E.

coli strains utilizes distinct approach for binding to the host cell and its disruption.

Evolution of diverse pathogens

Acquisition of foreign genetic materials (transposons and plasmids) played a key role in

reshaping genome of pathogenic microbes. HGT is one of the efficiently used


26

mechanisms that rapidly disseminate new traits to recipient organisms. Consequent loss

and gain of mobile genetic elements ensures the survival of the pathogenic microbes, in

adaptation to their host. LEE encoded pathogenicity islands (PAIs), is present on extra

chromosomal plasmids or incorporated into the chromosome of pathogens. PAIs are

bordered by insertion sequences or transposons, which are frequently inserted in the

nearer tRNA genes. E. coli characteristics virulence factors are primarily located on

PAIs, plasmids and other prophages. Although most of the prophages are defective in

nature, but still some can form infectious particles (Asadulghani et al., 2009). Acquired

traits through HGT, cannot only enable the recipient bacterium to explore new niches,

but also give them a chance to survival under these pressures. Acquisition of multiple

mobile genetic elements can make the recipient bacteria prone to new selective pressures

that could lead to the selection of such traits resulting in the creation of more virulent

organisms such as EHEC and EIEC (Wirth et al., 2006). It is worth mentioning, that this

evolution of pathovars through HGT might not always follow lineage specific mode; for

example, rest of the E. coli pathotypes (Ogura et al., 2009) incorporated EHEC virulence

genes (stx1, stx2, and stx2 variants) differently. The genome size of the E. coli

pathotypes is about 1 Mb greater than those of other normal flora organisms, primarily

due to frequent exchange of PAIs and other genetic material. Complete genome of E.

coli strains are contains about 2,200 genes and a pan-genome of around 13,000 genes

(Rasko et al., 2008). Interestingly, core genomes of pathogenic E. coli strains contain

about 5,000 genes, where 40-45% of them can make up the core genome. EPEC core

genome contains about 400 extra genes compared to E. coli K-12 sub strains, and 650

less genes than EHEC O157:H7 and 770 less genes than UPEC str. CFT073 (Iguchi et

al., 2009). Among E. coli pathotypes, EIEC has experienced more patho-adaptation due

to sequential loss of genetic material. Acquisition of the pINV plasmid give invasive


27

nature to the EIEC; whereas, a loss in the lysine decarboxylase gene region of the

genome containing cadA gene further enable to the adaption of an intracellular lifestyle.

Biphasic lifestyle of E. coli

Evolutionary scale of E. coli strains is consisted of host-dependent and independent

phases. E. coli strains and host interactions vary in complexity among different E. coli

pathotypes and host cells. Difference in the mode of pathogenicity indicated gradual

evolution of virulence factors on the genomic islands. Constantly evolving genomic

structure enabled the pathogens to invade diverse range of new hosts and niches. For

instance, pathogenic E. coli species upon entry into the host stomach via contaminated

food confront stressful environment due to the low pH in the intestinal tract. After safe

arrival in the intestinal tract by crossing through stomach, found a bit suitable

environment for growth and reproduction.

In an open environmental conditions vary greatly with geographic position and seasons.

Generally, low level of available carbon/energy sources, optimum temperatures, toxic

versus anoxic conditions and variable, low to high osmolarity will keep the pathogen

challenging at each step. Keeping in view of these new challenges, some strains of E.

coli might have acquired major and minor changes in the core genome such as

siderophore- mediated iron uptake systems (Schuberth et al.,2004) or ABC transporters

for uptake of amino acids and sugars. It is therefore stated the diverse feeding nature of

E. coli enable it to survive in an open environments (Ihssen et al., 2007).


28

Biochemical Properties of E. coli

Strains of E. coli are gram-negative short rods (bacilli) however; cultures of E. coli that

are more than 24 hours old may appear as cocci when viewed under the microscope

(Bettelheim, 1994; Prescott et al.,, 1996).

Biochemical Properties of E. coli Species.

Catalase reaction: E. coli species uses two main enzymes namely catalase and hydrogen

peroxidase to assist the conversion of hydrogen peroxide and superoxide back into

diatomic oxygen and water (Soomro et al., 2002). The catalase test involves the addition

of hydrogen peroxide upon bacterial cells grown in slants or plates. Formation of gas

bubbles indicates positive result.

Breakdown of tryptophan: Strains of E. coli hydrolyse tryptophan to indole, pyruvic

acid and ammonia by the use of tryptophan synthase. The pyruvic acid is further broken

down to release extra energy converting ammonia into amino acids. 98% E. coli strains

are indole positive. For the identification of E. coli O157:H7, an indole test is always

recommended (Alexandre et al., 2000; Soomro et al., 2002). Indole test involves the use

of a tryptone broth tube inoculated with a 24 h fresh E. coli culture and incubated for 48

h at 37 °C. The production of a distinct red colored upper layer of the broth-culture upon

pouring of 0.2 - 0.3 ml of Kovacs' reagent.

Fermentation of simple sugars: E. coli species ferment simple sugars like glucose and

lactose resulting in the production of an acid and liberation of gaseous products. It is

difficult to distinguish E. coli O157:H7 from other E. coli species due to advent

fermentation. Unlike other E. coli pathotypes, E. coli O157:H7 do not ferment D-sorbitol


29

in 24 h at 37°C. The failure to ferment D-sorbitol is relatively rare among other strains of

E. coli and so is extremely useful in discrimination of E. coli O157:H7 (Feng et al.,

1998). Due to the existence of similar colony morphology, sorbitol negative strains are

further serologically confirmed with O157 and H7 antisera (Feng, 1995).

Citrate reduction: Utilization of citrate is another distinguished characteristic used for

differentiation among enterobacteriaceae species. A differential media i.e. Simmons

Citrate Agar medium carries citrate as a carbon source and ammonium salts as a nitrogen

sources. E. coli strains that metabolize citrate using the ammonium salts, releases

ammonia and thereby cause an increase in the pH of the medium (Prescott et al.,, 1996).

Bromo thymol blue is used as the indicator dye in the medium. E. coli are citrate

negative and when grown in Simmons Citrate Agar does not change the agar from green

to blue (Holt et al., 1994).

Motility test for E. coli: Microbial motility can be confirmed by observing motility agar

tubes (Shelton et al., 2006). If microbial strain is motile, it will spread out the line of

streak (Farmer, 1999; Ware et al., 2000). Growth of non-motile organisms only occurs

along the stab line. E. coli and more specifically serotype O157:H7 are highly mobile

and will show turbidity throughout the tube. This is due to the possession of motility

antigens such as the flagella antigen by these organisms (Odenholt-Tornqvist &

Bengtsson, 1994).


30

Serological identifications

Serological tests: Serological tests are used to identify the antigenic properties such as

the flagella H and the somatic O-antigens possessed by the E. coli pathotypes. For

instance, to identify E. coli O157:H7, their ability to agglutinate in O157 antiserum is

always tested using slide, tube or latex agglutination test (Rice et al., 1992). During this

test, it is important to perform the appropriate control for auto agglutination. E. coli

O157:H7 sometimes may give false positive agglutination results due to contamination

with other sorbitol negative species such as E. hermannii.

Methyl Umbelliferyl-β-D-Glucuronide (MUG) test for E. coli: There are certain

strains of E. coli that produce β-glucuronidase and are thus MUG positive, however,

most E. coli O157 strains are MUG negative (Thompson et al., 1990)

Antimicrobial resistance in microorganisms

The mechanisms of antimicrobial resistance are described by using four categories:

„Bypass‟ is based on the structural nature of micro-organism‟s outer membrane

which is considered to be a barrier for drug‟s entry into the cell. The outer cell

wall makes these bacteria are resistant to many antimicrobials (Nikaidou et al.,

1994).

Enzymatic inactivation or modification of the antimicrobial. For example, by the

production of the β-lactamase enzymes in E. coli (Jacoby, 1994), which destroys

the β-lactam-ring‟s chemical structure of the penicillins; or, aminoglycoside-

modifying enzymes in Staphylococcus aureus (Shaw et al., 1985).

The organisms develop antimicrobial resistance through structural changes of the

drug target sites (for example, mutations in the genes lead to a structure of


31

changed drug target sites that inhibit drugs from binding to these target sites

(Webber and Piddock, 2001).

The efflux pump is a self-defense mechanism of a microorganism resulting in the

efflux of drugs that entered into the cell through cell membrane, before it can

contact the action site and exert its effect (Tenover, 2006). The efflux system is

considered a pump because the ejection process requires energy. This mechanism

exists in many situations of natural resistance of specific organisms and in many

kinds of antimicrobials such as tetracycline or fluoroquinolone (Van Bambeke et

al., 2006).

Antimicrobial resistance can be classified as either natural resistance or acquired

resistance (Todar, 2002). The natural resistance refers to an organism, which has the

inherent ability for resisting an antimicrobial. An example for this is the inherent

resistance of a Gram-negative bacterium like E. coli to penicillin G because there is no

reaction site of penicillin G in its structure (Kim et al., 2005). The acquired resistance

refers to a qualitative alteration of the genetic material of the organism as the result of

microbes changing in some ways to eliminate the effectiveness of drugs through

mutations. A mutation is a change in the DNA. That means, for example, that a mutation

in the gyrA gene of E. coli leads to the result that ciprofloxacin cannot bound an essential

bacterial enzyme required for the DNA replication. This allows E. coli to continue the

DNA replication in the environment with the presence of ciprofloxacin (Rodriguez-

Villalobos et al., 2005).


32

Antibiotic Resistance among Intestinal E. coli

E. coli has often-higher degrees of antimicrobials, which have a long history of use. E.

coli whether isolated from animals and humans showing resistance to antimicrobials

used in animals would also be resistant to antimicrobials used in humans (Miles et al.,

2006). E. coli strains isolated from domestic cattle and patients showed resistance to a

large group of antimicrobial agents tested (neomycin, gentamicin, sulphonamides,

chloramphenicol, ofloxacin, tetracycline, ampicillin, cephalothin, trimethoprim-

sulfamethoxazole, nalidixic acid, nitrofurantoin, and sulfisoxazole) compared with

isolates from human excretions, wildlife and surface water (Sayah et al., 2005). E. coli

often carries multi-resistant plasmids and it is considered as a reservoir of resistant genes

to transfer those plasmids to other species as well as pathogens in humans and animals

(Sorum and Sunde, 2001).

E. coli confers resistance to aminoglycosides by enzymatic modifications. Three classes

of modifying enzymes are responsible for aminoglycoside resistance. They include the

acetyltranferase, phosphotransferase, and adenyltransferase. Because of these

modifications, the binding affinity of the drug to ribosomes is altered, resulting in

resistance. In addition intrinsic and adaptive resistance that results in decreased uptake

have also been found in aminoglycoside-resistant Gram-negative bacteria (Levy, 2002).

The major mechanism of resistance to chloramphenicol is through enzymatic

modification by chloramphenicol acetyltranferase, using acetyl-coenzyme A as the acyl

donor to eventually convert chloramphenicol to 1, 3-diacetoxychloramphinicol. The

product then loses its ability to bind to the peptidyltransferase component of the 50S

ribosomal subunit rendering the drug inactive (Shaw et al., 1985). Another mechanism is

chromosomal mutation, which causes a lack of the entry sodium and potassium ions


33

leading to membrane impermeability to the drug (Toro, Lobos, Calderon, Rodriguez, &

Mora, 1990).

Antibiotics are often used therapeutically and prophylactically to treat human and animal

infections and in addition used as growth promoters in animal production (Bruinsma,

Stobberingh, de Smet, & van den Bogaard, 2003).

Several studies have reported significant increase in the number of antimicrobial resistant

E. coli O157:H7 strains from clinical, environmental and food origins. Studies have

reported emerging resistance against cephalothin, sulphatriad, colistin sulphate,

sulfamethoxazole and tetracycline (Magwira, Gashe, & Collison, 2005). The link

between use of antibiotics and development of bacterial resistance is well documented

(Hendriksen, Mevius, Schroeter, Teale, Jouy, et al., 2008). Use of antibiotics in farming

industry has played a pivotal role in the emergence and dissemination of antibiotic-

resistant bacterial strains (Barza and Travers, 2002). It has been reported that application

of antibiotics in animals meant for food may facilitate the development of resistant genes

in pathogens infecting these animals, which may eventually be transferred to human

through the foods (Hendriksen, Mevius, Schroeter, Teale, Meunier, et al., 2008).

Different antibiotic resistance profiles have been detected in E. coli O157:H7 strains of

humans, animals and foods origin (You et al., 2006).

Chemotherapy should not be prescribed for the treatment of human infection with E. coli

O157: H7 that could result in the release of more potent toxins in gut (Zhao et al., 2001).

E. coli O157:H7 infections in human mainly occur due to the ingestion of fecaly-

contaminated water, meat sources and vegetables. Determination of E. coli O157: H7

origin is required for the development of antimicrobial resistance profile. Antimicrobial

resistance is emerging due to the frequent exchange of mobile genetic elements among

commensals and pathogenic strains. The extensive use of antibiotics in both human


34

medicine and animal agriculture is suspected to have leads to a widespread dissemination

of antibiotic resistant genes (Callaway et al., 2003).

Different pathotypes of E. coli

Enteropathogenic E. coli

In developing countries like Pakistan, infantile diarrhea is caused by EPEC with high

mortality rate. Primarily EPEC form attaching and effacing (A/E) lesions on the host

brush border epithelial cells. The attached bacteria then erode the essential microvilli of

host cell to disrupt its actin by creating a characteristic patches below the site of

attachment. This characteristic in EPEC is brought about by eae genes encrypted on a 35

kb PAI (Pathogenicity Island) known as the locus of enterocyte effacement (LEE)

(McDaniel et al., 1995). The LEE activates T3SS that transfer bacterial toxin to the host

cell cytoplasm. Apart from LEE other non-LEE encrypted (Nle) effectors proteins also

their role in the attachment to the host cell (Deng et al., 2004).

Mechanism: EPEC, s binding to the host small intestinal enterocytes is believed to be

facilitated by attachment factor (EAF) plasmid encoded bundle-forming pili (bfp).

Structurally bfp are coiled rope-like fimbriae extension, which not only brought EPEC

strains close together for localized adherence, but also interact with N-acetyl-

lactosamine-containing receptors on host cell surfaces (Hyland et al., 2008). EPEC

attachment to the host cell is brought about by intimin and the translocated intimin

receptor (Tir). Attachment strategy of EPEC includes induction ofT3SS to rapidly shift

Tir into the hot cell cytoplasm through Ca2+ sensing. Afterward Tir showed on the

epithelial cells of host (Kenny, Abe et al., 1997) acts as an intimin receptor. Tir

interactions to the intimin lead to tir recruitment, which is then phosphorylated by

various host tyrosine kinases (18-20). Tir Phosphorylation brings Nck to the binding site,


35

thereby activating neural wiskott–Aldrich syndrome protein (N-wASP) and the actin-

related protein 2/3 (ARP2/3) complex to rearrange actin and form characteristics pedestal

(Kalman et al., 1999).

Enterohaemorrheagic E. coli

During the past two decades, EHEC gained considerable importance by causing

numerous foodborne gastroenteritis outbreaks. EHEC inhibit distal part of the large

intestine in humans and cause bloody diarrhea that may leads to hemorrhagic colitis

(HC) and hemolytic uremic syndrome (HUS) (Kaper et al., 2004). Transmission cycle of

EHEC from cattle to the humans may be completed via ingestion of fecaly contaminated

food and drinking water. Foodborne outbreaks due to EHEC serotype O157:H7 were

reported from North America, japan and parts of Europe. EHEC O157:H7 isolates

contain a 92 kb pO157virulence plasmid, having about 100 ORFs and encodes effector

proteins. However, characteristics virulence genes of O157:H7 is the shiga toxin

(Asadulghani et al., 2009; Nataro and Kaper, 1998).

Mechanism: Shiga toxin is further divided into Shiga toxin1, 2, which are encoded alone

or together in EHEC isolates. Among the subgroups Stx2 is more potent in causing HC

and HUS than stx1 (Nataro and Kaper, 1998). Shiga toxin is an AB5 type toxin, where B

subunit (pentamer) is non-covalently bound to an enzymatically active A subunit. There

exists no secretary mechanism for the release of shiga toxin in EHEC, therefore stx

transport is achieved through lambdoid phage-which initiate apoptosis in response to

DNA breakdown and SOS induction (Toshima et al., 2007). It is therefore, antibiotic

therapy should not be adopted, and as in response to the DNA damage more severe toxin

would be produced. Globotriaosylceramides (Gb3s), key stx receptors are present only

on Paneth cells of the intestinal and kidney epithelial cells (Toshima et al., 2007). Cattle


36

lack gb3s receptors in their gastrointestinal mucosa, and thereby remain asymptomatic

because of EHEC inability to colonize in cattle. The shiga toxin pentamer (stx B) part

attached with Gb3 receptor and cause membrane disruption to help in toxin entry. Once

entered shiga toxin is passed, golgi bodies vi endosomes for processing, where the shiga

toxin A part is enzymatically activated leading to apoptosis and cellular death.

Physiological role of shiga toxin attached to the underlying paneth cells is yet to be

explored. Interestingly macropinocytosis ingestion also facilitates the presence of stx in

Gb3-negative human intestinal cells. Inside these gb3 negative cells, stx does not prevent

protein synthesis nor induce apoptosis rather it is believed to suppress inflammatory

responses by inhibiting chemokine expression (Gobert et al., 2007).

Enterotoxingenic E. coli

ETEC is commonly associated with Travellers‟ diarrhea, and can cause severe

dehydration in children less than 5 years, where it ends up with devastating fatal

consequences. ETEC attachment to the host epithelial cells is brought about by

colonization factors (CFs), which are fimbrial, non-fimbrial, helical or fibrillar in nature.

Both CFs and flagella attach ETEC to the host epithelial cells. The outer-membrane

proteins Tia and TibA (Turner et al., 2006) aid specifically ETEC intimate attachment to

the host cell.

Mechanism: ETEC induced diarrheal infection occur due to the release of heat-stable

(STs) and heat-labile enterotoxin (LT) or both. Heat-stable enterotoxins STs are small

sized toxins categorized into STa or STb. STa subgroup is composed of 72-amino-acid

precursors, whereas STb consists of 48 amino acids. STa is primarily responsible for

human disease, by binding to the guanylyl cyclase receptors on epithelial cells of the

intestine. This simulation results in elevation of intracellular levels of cyclic GMP which


37

in turn activates the cystic fibrosis transmembrane conductance regulator (CFTR),

resulting in impaired absorption of Na+ and H2O efflux into the lumen (Turner et al.,

2006). Structurally heat labile AB5 toxin is identical to cholera toxin. The B subunit of

LT interacts with the monosialoganglioside GM1 on host cells; entry of toxin is marked

at lipid rafts (Kesty et al., 2004).

Enteroinvasive E. coli

It is commonly believed that EIEC and Shigella belong to the same class, by sharing

similar mode of pathogenicity. Virulence is mainly attributed to a 220 kb plasmid that

encodes a T3SS that is required for invasion (Ogawa et al., 2008).

Enteroaggregative E. coli EAEC are the second most common pathogen of Traveller‟s

diarrhea headed only by ETEC in both developed and developing countries. EAEC

always cause watery diarrhea that could contain small amount of mucus or blood. EAEC

primarily attached to the intestinal epithelial cells, by causing mild colon inflammation

(Nataro and Kaper, 1998). Characteristics disease pattern of EAEC is its stacked brick

layout of attachment to HEp-2 cell and virulence genes are present on 100 kb pAA

plasmids that carried genes for the synthesis of the aggregative adherence fimbriae

(AAFs), and initiate attachment of EAEC to the intestinal mucosa (Nataro and Kaper,

1998).

Diffusely Adherent E. coli

DAEC is a heterogeneous class, which utilizes characteristics attachment design on a

layer of HeLa and HEp-2 cells. Adherence design is facilitated by a group of proteins

expressed by a cluster of similar operons, consisting of both fimbrial (Dr and F1845) and


38

afimbrial (Afa) adhesins. DAEC pathotypes, which showed any of the Afa–Dr

adhesions, inhabit the small intestine thereby causing diarrheal infection in young

individuals from 18 months to 5 years and adults. All Afa–Dr adhesions factors act upon

brush border epithelial cells along with decay-accelerating factor (DAF), present on the

surface of urinary tract epithelial cells. DAEC on binding to DAF rearrange all of its

underlying molecules. DAEC also activates a Ca2+ dependent cell-signaling pathway,

thereby causing an elongation and dissociation of brush border epithelial cells microvilli

via disruption of cytoskeleton molecule. Combinatorial association of Afa–Dr adhesions

regulates the secretion of IL-8 from enterocytes for the transport of polymorphonuclear

neutrophils (PMNs) across the mucosal epithelial layer.

Uropathogenic E. coli

About 80% of UTI infections are caused by UPEC, resulting in the cystitis in the urinary

bladder and acute pyelonephritis in the kidneys. UPEC travel all the way down to the

urinary tract from intestinal tract, using proteins and amino acids as a source of energy

for causing an infection. The ability of UPEC to rise against the gradient in the urinary

tract from the urethra to the urinary bladder and reach to the kidneys shows unique

mechanisms for organ tropism. This complex mode of pathogenesis is thightly regulated

by a combination of several virulence factors i.e. multiple pili, secreted toxins (Sat) and

vacuolating autotransporter toxin (Vat), multiple iron procurement systems and a

polysaccharide capsule.

Mechanism: Adhesion to the uroepithelium is the first target of UPEC after getting

entered. Attachment to the uroepithelium is caused by fimbrial adhesion H (FimH)

factor, found at the tip of type1 pili. FimH is attached to the α3 and β1 integrins and

thereby recruiting UPEC at the site of invasions. Attached bacteria are internalized


39

through local actin rearrangement and Rho family. On internalization, UPEC form

biofilm-like complexes called intracellular bacterial communities (IBCs) or pods.

E. coli O157:H7

E. coli is a facultative Gram-negative anaerobic bacillus (Yoon and Hovde, 2008) that

distinctly colonize distal part of intestinal tract of most living organism within hours of

birth. E. coli O157:H7 is frequently isolated from patients suffering bloody diarrhea,

hemorrhagic colitis and HUS (Benjamin and Datta, 1995; Kaper, 1998; Yoon and

Hovde, 2008). E. coli O26:H11 members of EHEC family may also hemorrhagic colitis

(Robins-Browne, 1995). Cattle serve as natural reservoir of E. coli O157:H7, abiding

within cecum and colon (Hussein and Sakuma, 2005), where 10-80% of all cattle are

colonized (Welinder-Olsson and Kaijser, 2005). Remarkably, cattle remains

asymptomatic when inhabited by E. coli O157:H7, except young calves might

experience diarrheal infection. E. coli O157:H7 inhabitation was found helpful to the

animals (Hussein and Sakuma, 2005). Human beings carry gb3 receptor and thereby

highly sensitive to E. coli O157:H7 infection (Moxley, 2004).

The History and Epidemiology of Enterohaemorrheagic E. coli (EHEC)

EHEC cause a sever gastroenteritis sometime also called hemorrhagic colitis

symptomized as with severe abdominal cramping along with bloody diarrhea (Riley et

al., 1983). Transmission of EHEC usually occurs through ingestion of contaminated food

i.e. such meet, vegetable and other sources. Initial outbreak 1982, due to EHEC in North

America, was declared due to be the consumption of contaminated hamburgers caused

by a pathogenic E. coli (Armstrong et al., 1996). On serotyping this strain revealed a

diverse nature by carrying O-antigen 157 and H7-antigen. E. coli pathotypes differ from


40

other recognized strains of E. coli by having an additional virulence factors.

Enterohemorrhagic E. coli (EHEC) was named after the mode of pathogenicity by

causing bloody diarrheal infection leading to HC or HUS (Armstrong et al., 1996).

Second EHEC outbreak was observed in 1993, declared as the largest E. coli outbreak to

date. EHEC is still considered as an emerging pathogen of great concern, although it was

initially isolated in 1977 (Yoon and Hovde, 2008) and its human pathogenic nature was

recognized 1982. EHEC gained considerable importance among foodborne pathogens by

causing numerous foodborne illnesses (Kaper, 1998; Jores et al., 2004). EHEC based

foodborne outbreaks were reported both from developed and under developed countries

i.e. North America, the United Kingdom, and Japan (Whitworth et al., 2008). Higher

prevalence of E. coli O157:H7 was reported by CDC in United States during 1999, with

4744 confirmed cases of toxicoinfections with annual increase i.e. 2004 and 2005 there

were 2,544 and 2,621 cases confirmed (Yoon and Hovde, 2008). Prevalence rate of

EHEC infection differs greatly with geographic position (Garmendia et al., 2005).

Biochemical characteristics of E. coli O157:H7

Biochemical characterization of E. coli O157:H7 involves the application of indole test,

methyl red-Voges-Proskauer (MRVP), citrate utilization and lysine decarboxylation tests

(Radu et al., 1998). API 20E test kits are extensively used for the identification of E. coli

O157:H7 (Guyon et al., 2001). The polyclonal nature of E. coli serotype O157:H7 has

expedited its phenotypic identification using specific O157 and H7 anti-sera (Feng,

1995).


41

Clinical presentation of E. coli O157:H7

E. coli strains get enter into gastrointestinal tract by ingesting contaminated food. Upon

entry E. coli O157:H7 can cause a toxicoinfection by the secretion of shiga toxin the host

Signs and symptoms of the infections begin to appear after the incubation period of 1 to

8 days whereas, in some cases remain asymptomatic or showed mild symptoms. EHEC

infection usually begin with abdominal cramping and followed by watery diarrhea, along

with nausea and vomiting that may progress into hemorrhagic colitis within 2-3 days

(Yoon and Hovde, 2008). As a result, severe abdominal cramps are observed along with

bloody diarrhea. However, chilling or no fever is another symptom of hemorrhagic

colitis. Fortunately this unbearable infection is self-limiting and do resolve within one

week. Antibiotic therapy shouldn‟t be adopted for the treatment of shiga toxin producing

E. coli infection, they could result in induced complication due to the secretion of more

potent toxins (Bielaszewska and Karch, 2005; Ceponis et al., 2005; Kesty et al., 2004).

Because of antibiotic higher concentration of shiga toxin can enter into the blood stream

that may result in renal damage and other systemic effects in infants or old populations

(Kaper, 1998). HC may lead to HUS, which cause renal disorder among young

individuals (Yoon and Hovde, 2008). (Orth et al., 2006) and the immune-compromised

ones. 5-10% of hemorrhagic colitis develops into HUS (Welinder-Olsson and Kaijser,

2005; Yoon and Hovde, 2008). HUS mortality rate is about 3-17% (Welinder-Olsson and

Kaijser, 2005), and survivals are left with renal disorders. HUS destroys renal endothelial

cells by the development of microangiopathic hemolytic anemia (Yoon and Hovde,

2008) along with destruction of RBCs, thrombocytopenia (Orth and Wurzner, 2006).

Secretion of shiga toxins subtypes may cause further kidney disorders (Kaper, 1998;

Orth and Wurzner, 2006). These cytotoxins inhibit protein synthesis in glomerular

endothelial cells around arteries/veins and thereby results in the coagulation of platelets


42

and fibrin in the bowman capsule. Such type of reaction clogs the decreases filtration rate

of bowman capsule total. Hemorrhagic colitis may result in other forms of HUS called as

thrombotic thrombocytopenic purpura (TTP) expressed by symptoms like fever,

headaches, convulsions, lethargy, and encephalopathy (Yoon and Hovde, 2008).

Disease Burden of E. coli O157:H7

In the United States only, 350 foodborne outbreaks were reported during1982 to 2002,

registering about 8,600 cases of E. coli O157 infection (Rangel et al., 2005). Among

these, there were 17.4% (1,493) hospitalizations, 4.1% (354) cases developed HUS, and

0.5% deaths were reported. In the United States disease burden E. coli infection is about

73,500 illnesses, consisting of 2,168 hospitalizations and 61 deaths (Mead, 2000). In

2005, E. coli O157:H7 outbreak was reported in Oklahoma and East Scotland.

Virulence Genes of E. coli O157:H7

Virulence profile of E. coli O157:H7 are attributed to the activities of three cardinal

virulence factors. E. coli O157:H7 is severe pathogenic organism with an infectious dose

of about 1-100 CFU/mL (Karmali, 1989; Phillips and Frankel, 2000). Pathogenicity of E.

coli O157:H7 is due to the secretion of phage-encoded shiga toxins called Stx1 and Stx2

(Orth and Wurzner, 2006), passionately affecting eukaryotic cells (Riley et al., 1987).

Both stx1 and stx2 consist of enzymatically active subunit A (single) and 5 identical B

(binding subunits). Based on the structure they are classified into AB5 family of toxin.

The stx B subunit attached to globotriaosylceramide (Gb3) receptors present on the

surface of host endothelial and epithelial cells (Jores et al., 2004), whereas A subunit is

internalized into the host cell using endocytosis process, leading to the inhibition of

protein synthesis, by exerting its N glycosidase activity. Stx2 is more virulent than other


43

of E. coli toxins (Bielaszewska and Karch, 2005). The only difference between the shiga

toxins of E. coli O57:H7 and Shigella dysenteriae is by single amino acid.

E. coli O157:H7 can secrete stx1 and stx2 alone or together (Karmali, 1989). Although

secretion of stx2 alone indicates more pathogenic nature than in any, other forms (Li and

Hovde, 2007). Variants forms of shiga toxin include stx1, stx1c, stx1d, stx2, stx2c, stx2d,

stx2 deactivatable, stx2e, and stx2f (Jores et al., 2004).

The locus of enterocyte effacement (LEE) is a termed as bacterial pathogenicity island,

(PAI), a designated DNA segment, for the expression of specialized virulence factors

(Jores et al., 2004). Shiga toxin is activated and transported to the site of infection using

a combinatorial effect of LEE encoded factors consists of T3SS, intimin, translocated

intimin receptor (Tir), and other associated of effector proteins (De Grado et al., 1999).

Attaching and effacing lesion is attributed to a coordinated response of TTSS, a multi-

subunit organelle encoded within the LEE, assembled to form functional apparatus (Jerse

et al., 1990). Host cell membrane is invaded by creating a pore using this apparatus,

through which bacterial effector proteins are transported across to the host cell. Apart

from LEE encoded pathogenicity island (PAI), pO157 plasmid significantly aids in the

pathogenicity of E. coli O157:H7 (Lim et al., 2007). The pO157 plasmid contains about

100 open reading frames (ORFs) and is about 90kb (Yoon and Hovde, 2008).

Action of Shiga Toxins of E. coli O157:H7

E. coli O157 causes HUS and HC: H7 by the secretion of shiga toxins (Stx1 and Stx2),

due to attaching and evasion of vascular epithelial cells (Donnenberg and Whittam,

2001). The cytotoxic effect of stx on epithelial cells is due to the localized evasion of

host cell villus (Nataro et al., 1998). Both stx1 and stx2 consists of enzymatically active

A subunit non-covalently bonded to the pentamer B subunits which binds to gb3


44

receptors and other glycolipids on the host cells (Perna et al., 2001). The A part is

internalized by endocytosis and reached to the endoplasmic reticulum of the host‟s cell

(Donnenberg and Whittam, 2001). It is believed that the toxin binds at a specific target

on the 28S rRNA, which is A depurated adenine residue, inhibiting protein synthesis and

induce apoptosis. Stx receptors are located on endothelial cells and renal micro-vascular

endothelial cells are particularly sensitive to the toxin.

Transmission Routes of E. coli O157:H7 to Human

Water

The 2010 flood has affected about 78 districts in Pakistan, affecting 21 million people.


areas in a horrible situation. According to World Health Organization (WHO) 2010


consultations, where 708, 891 (13%) were made for acute diarrhea, followed by acute

respiratory infections (15%) 802, 670, (18%) 986,843 were for skin disease and (3%)

182, 762 were for malaria. Around 500, 000 pregnant women were among the affected

population. This calamity left long lasting impressions on the health of millions of

affected people. During this natural disaster, fecal contaminants were added to the water

bodies by all means, as if untreated or poorly treated sewage water, direct leakage of

septic tanks and spillage from sanitary passages.

Humans and animals are mainly responsible for contamination of both fresh and stored

water bodies by shedding pathogens through defecation in/near water channels or

catchment areas. They can also be transmitted through fruit and vegetables juices and

uncooked greens (lettuce and white radish sprouts (Cody et al., 1999). Waterborne

diarrheal outbreaks are attributed to the fecally contaminated water (Schets et al., 2005).


45

Recreational water sources can be also contaminated with these pathogroups (Verma et

al., 2007). Activities of these E. coli pathotypes resulting in an outbreak has been found

throughout the UK, USA, Canada, Japan, Sweden to name but a few (Woodward et al.,

2002; Cagney and Browning, 2004; Sartz et al., 2008; Uhlich et al., 2008).

Occurrence of E. coli pathogroups in the environment are supported by the

environmental conditions, soil texture etc. (Jiang et al., 2002). Although number of

foodborne disease outbreaks caused by E. coli pathogroups each year is low, but still

they are significant pathogens. E. coli pathogroups outbreaks are dedicated to the warmer

period of the year (July, August and September). Seasonality in prevalence of E. coli

pathogroups may be attributed to the increased housefly inhabitants during the summer

(Alam and Zurek, 2004), humidity and rainfall (Gagliardi and Karns, 2000) and waste

management (Rasmussen and Casey, 2001; Perencevich et al., 2008).

Meat and its Products

Prevalence of E. coli O157:H7 have been reported in animals, their carcasses, hides and

feces (Brichta-Harhay et al., 2007). Prevalence of E. coli O157:H7 is rarely studied in

Pakistan. Prevalence of E. coli O157:H7 in bovine products have been reported of about

8% -15.7% in cows (Wells et al., 1991; Chapman et al., 1997) and 1.8% in cattle

(Hancock et al., 1997). Proportion of animals infected with E. coli O157:H7 ranged from

0 - 60% (Blanco, et al., 1996). Higher prevalence of E. coli O157 has been reported in

cattle and their carcasses. However, they predicted that reduction in carcass prevalence

from pre-evisceration to post processing could be achieved if sanitary procedures are

strictly followed (Elder et al., 2000). There is seasonal variation in E. coli O157: H7

shedding in cattle. Maximum shedding of E. coli O157:H7 has reported during hot,

humid and rainy seasons (Edrington et al., 2006). However, no factors have been


46

identified, other than season that consistently affects the E. coli O157:H7 shedding rates

of cattle

Undercooked Ground Meat and its Products

Undercooked ground beef have been a kingpin in disease outbreaks of E. coli O157:H7

(Riley et al., 1983; Kassenborg et al., 2004). Epidemiological data indicate that the

prime risk foods of bovine origin are undercooked hamburgers (Reilly, 1998). Such

contaminated hamburgers or ground beef tend to possess a characteristic pink color at the

middle (Riley et al., 1983). Different foods that act as vehicles in E. coli O157:H7

outbreak are like processed foods such as yoghurt, cheese and fermented sausage have as

well been involved in food-borne outbreaks caused by E. coli O157:H7 (Gansheroff and

O'Brien 2000).

Milk and its Products

The contamination of milk by E. coli O157:H7 is often suspected to occur during the

milking process. Contamination of milk and products occur during its collection,

processing and handling. Dairy products have been implicated in E. coli O157:H7

disease outbreaks (Karmali et al., 1988). The bacteria have been isolated from cheese

sandwiches (Kassenborg et al., 2004). This has been possible most probably because the

organism is acid tolerant and can grow under low acidic condition (Conner and Kotrola,

1995).


47

Fruits, Vegetables, and their Products

Plants provide habitats for many species of microorganisms such as bacteria, yeasts and

filamentous fungi (Lindow and Brandl, 2003). According to Zhang bacterial populations

on spinach and rape phyllosphere were larger compared to celery, broccoli, and

cauliflower (Zhang et al., 2010). Pathogenic organisms including E. coli O157:H7 and

Salmonella are transient residents of plants. To survive and grow in the plant

environment, human pathogens have to compete with indigenous members of plant

microbial communities for nutrition, energy and colonization on the host (Brandl, 2006).

The presence of human pathogens on edible plants can be a source of human illnesses.

Foodborne disease outbreaks occur due to ingestion of contaminated fruits and

vegetables. In the US, 21% of E. coli O157:H7 outbreaks were linked to vegetable

products from 1991 to 2002 (Rangel et al., 2005), and from 1996 to 2007, 33 outbreaks

were associated with Salmonella contaminated fruits and vegetables (Callaway et al.,

2003).

Vegetables may get contaminants during any stage of production. Potential pre-harvest

means of contamination include soil, feces, irrigation water, water used for pesticide

applications, dust, insects, inadequately composted manure, wild and domestic animals,

and human handling (Beuchat, 2002). Human pathogens can be recovered on harvested

products and many among them are consumed raw. In Mexico, 5.8% of vegetable

samples were found contaminated with Salmonella (Quiroz et al., 2009).This percentage

is higher in countries where adequate sanitation practices are not applied. For example,

up to 76% of vegetable samples were found to be E. coli-positive in Vietnam.

Undercooked ground beef and unpasteurized dairy products are more responsible for the

foodborne disease outbreaks in the developing countries. However, vegetables, such as

cabbage, celery, coriander, cress sprouts, radish sprouts, alfalfa sprouts, lettuce, spinach,


48

and apple cider, have been increasingly associated with infection by E. coli O157:H7.

The feces of carrier animals can contaminate vegetables, because STEC isolates are

found as part of their normal intestinal flora. Most recently, a large outbreak occurred in

Europe where German scientists found the E. coli strains that caused 46 deaths and more

than 3900 illnesses in bean sprouts. In 2005, a large outbreak involving E. coli O157:H7

was associated with lettuce. One hundred and thirty-five cases were confirmed, including

11 cases of hemolytic uremic syndrome. All these cases were infected by the E. coli

strain after consumption of lettuce in contaminated water (Soderstrom et al., 2008). In

2006, two outbreaks of foodborne illness caused by E. coli occurred back to back in the

US.

E. coli O157:H7 foodborne outbreaks are caused by fresh fruits and ready to eat salads

containing tomatoes, lettuce, cucumber, onions and carrots, especially those used in

salads (Brooks et al., 2004; Mukherjee et al., 2004). Sometimes these outbreaks may

occur due to the consumption of fruit juices like apple (Cody et al., 1999). Presence of E.

coli O157:H7 on vegetables and fruits are believed due to be the use of cattle manure or

use of potentially contaminated water with E. coli O157:H7 for irrigation (Solomon et

al., 2002; Johannessen et al., 2005). Vegetable associated diseases outbreaks are

attributed to the consumption of leafy lettuce (Ackers et al., 1998), potatoes (Chapman et

al., 1997), radish sprouts (Michino et al., 1999; Watanabe et al., 1999) and alfalfa

sprouts (Banatvala et al., 2001). Vegetable may be contaminated during processing like

cutting and slicing (Cassin et al., 1998). The cases of E. coli O157:H7 on vegetables

have been common mostly in salad bars (Brooks et al., 2004).


49

Control of the spread of E. coli O157:H7 and protection of public health

Prevention of food and water is a key approach to eliminate or avoid food borne E. coli

O157:H7 outbreak is in both developed and underdeveloped countries.

Protection of Water Sources

Water usually is contaminated by the addition of fecal wastes of both human and animal

origin. Poor sanitation can result are numerous disease outbreaks like that of E. coli

O157:H7. In developing countries, the use of untreated or poorly protected water for

drinking purpose have always been linked to acute diarrheal diseases. Combination of

multiple treatment processes must be adapted to for maintaining hygienic status of the

drinking water.

Control of E. coli O157:H7 Foodborne Outbreaks

Foodborne disease outbreaks are attributed to the food items obtained from unsafe

sources, contaminated raw food items, poor food storage, and improper personal hygiene

status during food preparation. Contamination may also occur due to the inadequate

cleanliness of kitchen, equipment and utensils. Undercooking may also lead to the food

contamination, cooling and reheating of raw food and prolonged time lapse between

cooking and eating of the foods. All these factors are responsible for numerous outbreaks

in both developed and developing countries (Kassenborg et al., 2004; Rangel et al.,

2005).

On Farm Control

Control measures must be taken at the dairy farms and nursery homes, since it is evident

that cattle are the primary reservoir of E. coli O157:H7 (Lejeune et al., 2004). Diet


50

management has also been reported to be helpful in controlling the prevalence of E. coli

O157:H7 in cattle.

Use of Antibiotics

The use of antibiotics in the farm animals as a growth promoters and yield enhancement

are under strong criticism, which is will continue in the near future (Callaway et al.,

2003; Panos et al., 2006). Extensive ad misuse of antibiotics as a therapeutic agent and in

agriculture has led to the wide spread transmission of antibiotic resistance genes in the

target pathogens through HGT (van den Bogaard and Stobberingh 1999).

Vaccination

Vaccination can play a crucial role in the prevention of infection and gave one a better

chance live. Immunization can greatly reduce the disease burden of E. coli, by

eliminating pathogen shedding and colonization. Low shedding of E. coli O157:H7 by

vaccinated calves have been reported in Canada (Finlay, 2010).

Use of Bacteriophages

Role of shiga toxin producing bacteriophages and their role in controlling E. coli O157

groups of researchers (Morita et al., 2002; Fischer et al., 2004; Tanji et al., 2004) have

tested H7 in vivo and in vitro. However, this phenomenon is not well documented in

farmhouse. Bacteriophage, as a vaccinating agent has shown some promising results

against E. coli O157:H7 in sheep (Bach et al., 2003).


51

Slaughtering and Dressing

Hides and skins serve as reservoirs of E. coli O157:H7 contaminating the carcasses

during slaughter (Heuvelink et al., 2001; Gun et al., 2003). It is therefore recommended

to wash the cattle and other animals before taken to the slaughter. Chemical washing of

the skin and carcass are recommended for the reduction of E. coli O157:H7 population.

Animal slurry and manure should be properly disposed, which prevents contamination of

water supplies or ready-to-eat fruit and vegetables (Nicholson et al., 2005).

Chapter 3 Materials and Methods

52

Materials and Methods

The present research work was conducted during January 2010 to December 2012.

Diarrheal stool samples were collected from different wards (medical, ICU and OPDs) of

the study area Hospitals and flood affected refugee camps in southern parts (Bannu,

Kohat, Lakki Marwat and D. I. Khan) of Khyber Pakhtunkhawa, Pakistan. These clinical

samples were transported to Microbiology Research Laboratory and processed for the

isolation/identification of diarrhaegenic E. coli pathotypes including E. coli O157:H7

serotype. E. coli strains were identified according to standard microbial identification

methods. Antimicrobial sensitivity testing and confirmation of E. coli O157:H7 and its

molecular characterization were performed according to the standard protocols.

Epidemiology

Patients/Clinical Data Collection

Information regarding age, gender, sample source (stool) and nature/status of patient

(indoor patient/outdoor patient) was recorded for each sample on a separate proforma.

Transport of Samples

Samples were collected in wide mouth sterile container, sealed in sterile bags and

transported to the laboratory in carry Blair medium at optimum temperature.

Statistical Analysis

To statistically analyze the data, SPSS Statistics 16.0 software (SPSS Inc, Chicago) for

Windows was used. For all categories, variables were reported in numbers and

percentages. Association of the variables was analyzed using Chi-Square (X2) test. P

value less than 0.05 was defined as statistically significant.


53

Sample Processing and Identification of Culture Isolates

Bacterial Strains

Quality control was maintained at each step of isolation and identification. E. coli HB101

was used for quality control of the Gram‟s stain, biochemical tests, media preparation,

antibiotic susceptibility testing.

Isolation and Biochemical Identification of DEC Pathogens

About 50 ml of water sample was passed through a 0.45-µm filter paper and inoculated

in 10 ml of E. coli broth (EC broth) at 37°C for 4 h. 20 µg/ml novobiocin was added, and

incubated at 42°C for 24 h. A 50 µl aliquot of the enriched culture was poured onto both

Sorbitol MacConkey agar (SMAC; Oxoid) and Cefixime-Tellurite-Sorbitol MacConkey

agar (CTSMAC; Oxoid) and incubated at 42°C for 24-48 h. Identification of bacterial

isolates was made through conventional microbiological tests, including colony

morphology, Gram‟s staining and biochemical characteristics. Suspected colonies (clear

to smoky grey) were tested for the O157 antigen by latex agglutination (E. coli O157

Dryspot test; Oxoid, USA).

Biochemical Identification of Isolates

All isolates were identified through standard biochemical tests including; triple sugar

iron test, indole production test, methyl red test, Voges-Proskauer test, citrate utilization

test, motility testing and urease test.

Preparation of 0.5 McFarland Standards

To prepare 1.75% w/v solution of BaCl2, 2.35 g of dehydrated barium chloride was

dissolved in 200 ml of distilled water. Similarly, 1 ml of concentrated sulfuric acid was


54

mixed with 99 ml of distilled water to prepare (1% v/v) solution of sulfuric acid. Added

0.5 ml of BaCl2.2H2O solution to 99.5 ml of sulfuric acid solution to make 0.5%

turbidity standard and stirred constantly. The suspension was thoroughly mixed to make

sure that it is homogenous. Matched cuvettes were used to measure absorbance at a

wavelength of 625 nm using a spectrophotometer with a 1 cm light path. Water was used

as a blank. The acceptable absorbance for 0.5 McFarland standards was 0.08-0.13

Disc Diffusion Method

Overnight fresh cultures were used to make lawns on Mueller-Hinton agar (MHA)

(Difco BD, France). The inoculum was prepared by making a direct saline suspension of

isolated colonies selected from an 18 to 24 hour culture. The suspension was adjusted to

match the 0.5 McFarland‟s turbidity standard, using saline and a vortex mixer. Cotton

swab was dipped into the culture suspension. Culture was mounted on Mueller-Hinton

agar plate. The plate was allowed to dry for 5 minutes. The antibiotic discs were

bestowed onto Mueller-Hinton agar plate. No more than 5 discs were placed on one 150

mm plate or more than 8 discs on a 90 mm plate using modified Kirby-Bauer method.

The plates were incubated at 35°C for 16 to 18 hours at optimum temperature. E. coli

HB101 was used as control. Inhibition zones were measured in millimeters (mm), and

the isolates were classified as “resistant”, “intermediate” and “sensitive” according to

clinical laboratory standard institutes criteria (CLSI, 2006).

Isolation of Bacteria from Stool

Samples were inoculated directly into sterilized LB broth for culture enumeration. A

loop from the sample was inoculated into 5ml LB broth tube, which was incubated at


55

37°C for 24 h for culture enrichment. After successful culture enrichment, overnight

grown culture was subjected to identification.

Culture Identification:

Phenotypic and colony morphology based identification was carried out by growing the

isolated culture on differential growth media. E. coli serotype produced characteristic

pink colonies on media having sorbitol and β- glucocurinidase (β GUD). Based on this

property, differential media were used for preliminary identification. Overnight grown

cultures were screened by inoculating them on (1) MacConkey agar, (2) Eosin

Methylene blue agar (EMB), (3) Sorbitol MacConkey agar and (4) Cefixime Tellurite

Sorbitol MacConkey Agar and incubated at 37ºC for 24 hours aerobically.

Colony Morphology

Isolates were identified because of colony morphology on nutrient agar medium.

Characteristic features, observed on the different respective media, include; size (large,

moderate, small, pinpoint), pigmentation (color of colony), form (irregular, circular,

rhizoid), margins (entire, undulate, lobate, serrate, filamentous) elevation (flat, raised,

convex).

Procedure of Gram’s staining:

A drop of distilled water was taken on the center of a clean glass slide and a colony was

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

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

flame for two or three times. Smear was covered with crystal violet stain for 30-45

seconds. Then stain was washed with distilled water and covered with gram‟s iodine for


56

30-45 seconds. Again slide was washed with distilled water and decolorized with 70%

ethanol and wash with distilled water. Finally, the smear was stained with safranin

(counter stain) and smear was allowed to air dry. Test specimen was examined and

compared with positive and negative controls microscopically, under 100x using

immersion oil (Cheesbrough, 2011).

Identification of E. coli O157:H7

The overnight grown cultures were identified based on colony morphology. Suspected

colonies were transferred to Eosin Methylene Blue Agar plates where presumptive E.

coli culture produces a characteristic green metallic sheen. Based on the observation of

metallic sheen preliminarily identified E. coli was inoculated on MacConkey agar, where

they produce light pink colonies. Other pathogens like Salmonella and Shigella produced

waxy and raised colonies somewhat reddish in color. Suspected colonies were grown E.

cefixime (0.05 mg/l) and potassium tellurite (2.5 mg/l)-Sorbitol MacConkey agar (CT-

SMAC, Merck, SA) (Dynal product brochure, 2006).

Serodiagnosois of E. coli O157: H7

The Oxidase positive Gram negative, and indole positive were further subjected to

serotyping with DrySpot E. coli O157 kit.

Glycerol Preservation of Culture Strains

For long-term preservation, E. coli strains were inoculated in nutrient broth and

incubated at 37°C for 24 h. After incubation, 200 μl of the liquid culture was transferred

to 5 ml of LB broth and incubated for 5 h at 37°C. After 5 h incubation, liquid culture

was centrifuged at 5000 rpm for 2 min to harvest the cells.


57

Fig.1. Study flow chart

Vegetables &

its products

Meat & its

products

Pond water

Normal

faeces

Diarrheal

faeces

Cattle

faeces Diarrheal

Water

Antibiotic sensitivity test

Plasmid Extraction

Molecular Characterization of

eae, stx1, stx2, intimin, bfp gene

Serotyping

Identification of E. coli O157

E. coli isolation, single colony selection, and picking for further characterization

Drain water Tap

Biochemical

Sample collection

Stool

Sample

Feces

Sample

Food

Irrigattion water


58

Molecular characterization of Diarrheagenic E. coli O157:H7

DNA Extraction with QIAamp DNA Mini Prep Kit

Prepare an over bacterial culture in LB broth. Pipette out 1 ml of the culture suspension

into microcentrifuge tube and spun at 7500 rpm (5000 rcf) for 5 min. After

centrifugation, discarded the supernatant, and added 180 μl ATL buffer. Added 20 μl of

Proteinase K mixed by vortexing and incubated at 56°C for 3 hours (vortex 2-3times /hr)

during incubation. At the end, centrifuged it for 15 sec to remove the droplets. Added 20

μl of RNAase A (100 mg/ml), mixed it by vortex for 15°C. Incubated at room

temperature for 4 min. After incubation, vortex it again for 15 sec. Added 200 μl of AL

buffer, vortexed it for 15 sec, and incubated at 70°C for 10 min. Centrifuged it again for

15 sec. Add 200 μl Ethanol 96%, vortexed for 15 sec and centrifuged it for 5 sec.

Carefully applied the mixture along with the precipitates to the QIAamp mini spin

column. Closed the cap and centrifuged at 8000 rpm (6000 rcf) for 1 min. placed the

mini spin column in a 2 ml collecting tube.

DNA Quantification via Spectrophotometer (Beckman Coulter)

Bacterial DNA was quantified using Beckman spectrophotometer (USA), after 10 time

dilution (90 µl of sterile Milli-Q + 10 µl DNA).


59

Table 1. Bacterial DNA concentration (PA15, PA 33, PA 38)

PA 15 without dilution

PA 15 17.54 ug/ul

B 79.62 ug/ul

PA 33 35.63 ug/ul

B 62.89 ug/ul

PA 38 0.277 ug/ul

B 73.46 ug/ul

DNA Quantification via Nano drop

Bacterial DNA was quantified using Nano drop after 10-time dilution (90 µl of sterile

Milli-Q + 10 µl DNA). Add 1.5ul of mili Q water and blank it. Now add 1.5 ul measure

(PA4 1.3ug/ul *700) =910 by placing the cursor on DNA option.

Polymerase Chain Reaction (PCR)

Polymerase chain reaction (PCR) was performed to detect Shiga toxin producing genes

of E. coli O157:H7 (stx1, stx2 and stx2c), LEE encoded attaching and effacing lesions

(eae), Intimin translocating gene (tir), heamolysin gene (hlyA), and heat stable toxin (st)/

heat labile gene (lt) of Enterotoxigenic E. coli, bundle forming pili (bfpA) of EPEC.

Multiplex PCR Assay Condition.

Multiplex PCR Assay

Multiplex PCR assays were conducted using a reaction volume of 22 μl containing 5 ml

(20 pmol) of template DNA, 5 μl of (10X) PCR buffer, 5 μl of a Q solution (10X buffer),


60

0.25 μl of (250 U) of Taq-polymerase per ml (Sigma, USA) and 0.75 μl (20 mM) of each

primer (Sigma) and 7 μl of PCR water. PCR conditions set up in a Gene Amp PCR

system 9700 (AB Applied Biosystem) were as follows: 96°C for 5 min, 94°C for 30 sec,

57°C for 30 sec, and 72°C for 1 min in 35 cycles, with a final 5- min extension at 72°C.

Concentrated PCR products were diluted using Orange G dye. Diluted PCR products (10

μl) were confirmed by running on 2% (wt/vol) (Sigma) agarose gel electrophoresis. The

PCR products were visualized and photographed under UV light.

Gel Electrophoresis

PCR products and DNA samples were analyzed by gel electrophoresis using 2% agarose

gel , prepared by dissolving 1 g of agarose in 50 ml TAE 1X (50 x TAE buffer: 242 g/L

Tris, 18.61 g/L Na EDTA, 57 ml glacial acetic acid). Gel was prepared in an oven by

boiling the solution for 2 sec. About 5 μl of ethidium bromide was added to the solution

before pouring into the horizontal gel caster. For all samples, 5 μl loading dye, orange G

dye (30% v/v glycerol and 0.25% w/v each of bromophenol blue and xylene cyanol FF)

was used in 10% final concentration in the sample. Low mass DNA ladder was loaded as

size marker in the first well and gel was run at 120 V for 20+20 min (BioRad, USA).

After 20 minutes, observed for bands under UV light using UVP Bio-spectrum 300

imaging system (Bio-Rad, CA, US).


61

Table 2. Oligonucleotide sequence of primers used for the detection of shiga toxin

producing genes.

Shiga Toxin

primer

5' --> 3' primer Forward

(primer name)

5' --> 3' primer Reverse

(primer name)

Source Target/comment

Length

(bp)

stxAB2

(EDL933)

GGGTCTGGTGCTGATT

ACTTC

GTTACCCACATACCA

CGAATCAG

Kasper CW et

al., 1314

stx1 ATAAATCGCCATTCGT

TGACTAC

AGAACGCCCACTGAG

ATCATC

Rajkhowa et

al., 2010

180

stx2 GGCACTGTCTGAAACT

GCTCC

TCGCCAGTTATCTGA

CATTCTG

Rajkhowa et

al., 2010

target subunit A of stx2

and stx2c

255

stx2c

CTGAACAGAAAGTCA

CAGTYTTTA

GGCCACTTTTACTGTG

AATGTATC

Manning 2010

PLoSone

Targets region spanning

subunit A and B

182

stx2c

AGTACTCTTTTCCGGC

CACT (b)

GCGGTTTTATTTGCAT

TAGT (a)

Yukiko Asani

et al 2013

Targets Subunit B of

stx2c 124

stx2

TCCCGTCAACCTTCAC

TGTA (b)

GCGGTTTTATTTGCAT

TAGC (a)

Gehua Wang et

al 2002 Targets Subunit B of stx2 115

LSPA (Lineage Specific Polymorphism Assay)

Lineage specific polymorphism assay was performed according to the (Benson AK et al.,

2004) for the detection of six housekeeping genes fold sfmA, z5935, uhcG, rbsB, rtcB,

arp iclR.

Multiplex PCR for Lineage Specific Polymorphism Assays

For each reaction, 1 μl of template DNA was combined with 5 μl of PCR 5X buffer (20

mM Tris- HCl, pH 8.4, 50 mM KCl) (Invitrogen), 5 μl of Q solution, 0.75 μl of Taq

DNA Polymerase (Invitrogen), and a 1 μl of each forward and reverse primer for all six

markers. PCR conditions were 1 cycle at 94°C for 5 min; 94°C for 30 s, 50°C

(decreasing 1°C/cycle) for 45 s, and 72°C for 1 min; 20 cycles of 94°C for 30 s, 52°C for

45 s, and 72°C for 1 min; and 1 cycle at 72°C for 5 min.


62

Table 3. Oligonucleotide sequence of primers used for lineage specific polymorphism

Assay

PRIMER

(LSPA 6)

5' --> 3' primer

Forward

5' --> 3' primer

Reverse


Length

(bp)

foldDsmfA

TACGTAGGTCGAAG

GG

CCAGATTTACAACG

CC

Benson AK et

al 2004

lineage specific

polymorphism assay 161 or 170

Z5935 GTGTTCCCGGTATTT

G

CTCACTGGCGTAAC

CT

Benson AK et

al 2004

lineage specific

polymorphism assay

133 or 142

yhcG CTCTGCAAAAAACT

TACGCC

CAGGTGGTTGATCA

GCG

Benson AK et

al 2004

lineage specific

polymorphism assay

394 or 472

rbsB

AGTTTAATGTTCTTG

CCAGCC

ATTCACCGCTTTTTC

GCC

Benson AK et

al 2004

lineage specific

polymorphism assay

218 or 209

or 214

rtcB

GCGCCAGATCGATA

AAGTAAG

GCCGTTGTAAACGT

GATAAAG

Benson AK et

al 2004

lineage specific

polymorphism assay 270 or 279

arp-iclR

GCTCAATCTCATAAT

GCAGCC

CACGTATTACCGAT

GACCG

Benson AK et

al 2004

lineage specific

polymorphism assay

315 or 333

or 324

Table 4. Oligonucleotide sequence of primers used to detect eae, tir and hlyA genes

PRIM

ER

5' --> 3' primer

Forward

5' --> 3' primer

Reverse


Length

(bp)

eae GCCGGTAAAGCGA

CTGTTAG

ATTAGGCAACTCG

CCTCTGA

Manning 2010

PLoSone

inferes presence of the LEE island 138

Tir ACTTCCAGCCTTCG

TTCAGA

TTCTGGAACGCTT

CTTTCGT

Manning 2010

PLoSone

inferes presence of the LEE island 206

hly-A

CCAGGAGAAGAAG

TTAGAG

CAGACCATGTATC

CTTACC

Manning 2010

PLoSone

present on pO157 plasmid allows for

hemolysis activity

88


63

Table 5. Oligonucleotide sequence of primers used to detect shiga toxin bacteriophage

insertion sites in stx genes

PRIMER

NAME

5' --> 3' primer

Forward

5' --> 3' primer

Reverse length (bp) Target/comment

Length

(bp)

yehV

insertion site

region

AAGTGGCGTT

GCTTTGTGAT

(A)

AACAGATGTG

TGGTGAGTGT

CTG (B)

Besser et al.,

2007 and

Shaikh et al

2003

presence of stx1 prophage 340

yehV right

junction

AAGTGGCGTT

GCTTTGTGAT

(A)

GATGCACAAT

AGGCACTACG

C (primer E)

Besser et al.,

2007 and

Shaikh et al

2003

stx1 prophage detection 824

yehV left

junction

CACCGGAAG

GACAATTCAT

C (F)

AACAGATGTG

TGGTGAGTGT

CTG (B)

Besser et al.,

2007 and

Shaikh et al

2003


wrbA

insertion site

region

AGGAAGGTA

CGCATTTGAC

C (primer C)

CGAATCGCTA

CGGAATAGAG

A (D)

Besser et al.,

2007 and

Shaikh et al

2003


wrbA right

junction

AGGAAGGTA

CGCATTTGAC

C (primer C)

ATCGTTCGCA

AGAATCACAA

(primer G)

Besser et al.,

2007 and

Shaikh et al

2003


wrbA left

junction

CCGACCTTTG

TACGGATGTA

A (H)

CGAATCGCTA

CGGAATAGAG

A (D)

Besser et al.,

2007 and

Shaikh et al

2003


stx2

antiterminato

r Q gene

junction

CCGAAGAAA

AACCCAGTAA

CAG (595)

CGGAGGGGAT

TGTTGAAGGC

(Q933)

Besser et al

2007

presence of stx2 prophage:

ECH74115_3533 to ECH74115_3538

includes antitermination protein, stx

subunit A

967

stx2c

bacteriophage

O gene

ATGCGCAAGA

CATACGGATT

C (OF163)

TGCACAAACG

CCCTGACATA

(OR695)

Besser et al

2007

the presence of srx2c prophage:

ECH74115_2921 DNA replication protein

533

stx2c GGGCGCATGG ACTTCCCGTC Besser et al presence of srx2c prophage: 321


64

bacteriophage

Q gene

GTTTATTCA

(QF69)

GGCAGGTTG

(QR389)

2007 ECH74115_2910 late antiterminator

protein

argW phage

insertion site

(EDL933)

AACGACATGA

GCAACAAG

AGCCCTTAGG

AGGGGC

find author's

name


yehV forward

(EDL933)

AAGTGGCGTT

GCTTTGTGAT

AACAGATGTG

TGGTGAGTGT

CTG

Hauser 2013

49kb

(same

in

Sakai)

WrbA(EDL9

33)

GGTATTAACT

ACGCGGTAAG

CTCC

CCAGTACCGG

TGGAACTAAA

GAC

Kasper CW et

al.,

646

SbsB(EC411

5)

GTCTGGCATC

CTTCAAAGAC

AG

GACAATCTCT

TCCGCGTACT

G

Kasper CW et

al.,

1804

Chapter 4 Results

65

Results

The present study was conducted during March 2010 to September 2012 to detect the

prevalence and molecular characterization of diarrheagenic E. coli pathotypes in

Southern parts of Khyber Pakhtunkhawa, Pakistan.

During this study, 515 Diarrhaegenic E. coli strains were isolated from 900 clinical stool

specimens collected from different units of hospitals and outdoor patients in the area

under study. Of these 515 isolates, 64 were confirmed as Shiga toxin producing E. coli

O157:H7 serotype through latex agglutination tests and presence of stx1, or stx2 genes

using molecular techniques. These E. coli O157:H7, Enteropathogenic E. coli, and

Enterotoxigenic E. coli isolates were processed to detect the frequency of shiga toxin

producing genes (stx1, stx2, stx2c), eae, tir, hlyA, bacteriophage insertion sites

Bacterial Isolates Identification

All the 515 samples were identified through culture enrichment in LB broth then

culturing on (1) MacConkey agar, (2) Eosin Methylene blue agar (EMB), (3) Sorbitol

MacConkey agar and (4) Cefixime Tellurite Sorbitol MacConkey Agar for identification.

Diarrheagenic E. coli produced characteristic pink colored colonies on differential media

like Sorbitol-MacConkey agar, indicating lactose fermentation on MacConkey agar (Fig

4.1) and produced green metallic sheen on EMB agar. Gram-negative nature of DEC was

identified using Gram staining and conventional biochemical tests. For confirmation, six

standard biochemical tests were performed for every isolate of E. coli (Table 6). API E20

identification system was used for some of the isolates, which were not clearly identified

by conventional biochemical method.

Chapter 4 Results

66

(a)

(b) (c)

Fig 4.1: Microscopic examination of (a) Diarrheagenic E. coli on differential media (b)

Microscopic image of E. coli (c) Serotype O157:H7 growth on Sorbitol MacConkey agar

Table 6. Biochemical tests used for the identification of E. coli

Isolat

es

MacCkonkey

agar

Indole motility

Slope

TSI

But

t

Gas

H2S

citrat

e

Urease Oxidase

Lactose

Fermenting

colonies

ve

ve

Y

Y

-ve

-ve

-ve

-ve

-ve

Based on the results of these biochemical test isolates were confirmed as diarrhaegenic Escherichia

coli

Chapter 4 Results

67

Detection of E. coli O157:H7 among the Diarrheagenic E. coli Pathotypes

E. coli O157:H7 serotype was determined by their ability to produce characteristic

colorless colonies on cefixime-tellurite Sorbitol-MacCkonkey agar. Confirmation of

these isolate was made through E. coli O157 DrySpot kit (Oxoid, USA), where O157:H7

produced visible agglutination with antibodies specific for O157 antigens (Fig 4.2).

Fig 4.2: Serotype O157 positive agglutination test.

Sample Distribution

During the present study, 515 diarrheagenic E. coli strains were isolated of 900 clinical

stool samples examined for the prevalence of diarrheagenic E. coli, showed 57%

prevalence.

Fig 4.3. Overall distribution of diarrheagenic E. coli among the study group

900

515

0

200

400

600

800

1000

No of samples No of DEC isolated

No

of

sam

ple

s ex

amin

ed

No of DEC strains isolated

Series1

Chapter 4 Results

68

Based on stool physiology, 54.4% (n= 280) E. coli strains were isolated from watery

diarrheal samples, compared to 37.7 (n= 194) isolated from mucoid stool and 8% (n= 41)

from bloody diarrheal stool specimens as shown (Fig 4.4).

Fig 4.4. Prevalence of DEC strains in diarrheal categories

Age distribution of Diarrheagenic E. coli

The age of patients was categorized into seven groups; Group I (from one month up to

10 years), Group II (11-20 years), Group III (21-30 years), Group IV (31-40 years),

Group V (41-50 years), Group VI (51-60 years) and Group VI (61 years and above).

Number of samples collected from each respective age group and its percentage values

as: Group I (from one month up to 10 (n=227, 25%), Group 2 (11-20 years) (n=201,

22%), Group 3 (21-30 years) (n=126, 14%), Group 4 (31-40) (n=116, 12.9%), Group 5

(41-50) (n=90, 10%), Group 6 (51-60) (n=79, 8.8%) and 61 years and above (n=61,

6.8%).

280

194

41

0

50

100

150

200

250

300

Watery stool

(440)

Mucoid stool

(391)

Bloody stool

(64)

No

of

DEC

str

ain

s is

ola

ted

No and physical status of samples

No of E. coli Isolated

Chapter 4 Results

69

Of group I, 155 (30%) isolates were confirmed as E. coli strains. Similarly, number and

percentage isolation of DEC strains from each respective age group is as follows: Group

II 137 (26.6%), Group III 59 (11.4%), Group IV 47 (9.2%), Group V 44 (8.6%), Group

VI 42 (8.2%), and Group VII 31 (6%) (Fig 4.5). However, none of the age groups

achieved statistical significance.

Fig 4.5. Overall distribution of diarrheagenic E. coli among different age categories.

Key: Group I (from one month up to 10 years), Group II (11-20 years), Group III (21-30

years), Group IV (31-40 years), Group V (41-50 years), Group VI (51-60 years) and

Group VI (61 years and above).

155

137

59 47 44 42

31 30 26.6

11.4 9.2 8.6 8.2 6

0

20

40

60

80

100

120

140

160

180

Group I(227)

II(201)

III(126)

IV(116)

V(90)

VI(79)

VII(61)

No

& %

age

of

DEC

str

ain

s is

ola

tes

Age group (I-VII) and their respective no of stool samples

No of DECisolates Percent isolation

Chapter 4 Results

70

Gender Distribution

A higher percentage of E. coli isolates was reported from females as compared to male

(Table 3). Of 515 E. coli isolates, 206 (40%) were obtained from male patients and 309

(60%) from female patients (Fig 4.6).

Fig 4.6. Gender wise prevalence of diarrheagenic E. coli

40

60

0

10

20

30

40

50

60

70

Male Female

Per

cent

pre

val

ence

Gender

Chapter 4 Results

71

Ward Distribution

Most of the isolates were recovered from outdoor patients while 286 were obtained from

hospitals. The frequency of E. coli strains recovered different units of hospitals were as;

31.8% (n=91), 52% (n = 149), 3.15% (n = 9) and 12.94% (n = 37) from medical ward,

Pediatrics ward, OPD and ICU, respectively (Fig 4.7).

Fig 4.7. Percentage distribution of diarrheagenic E. coli based on sample origin

31.8

52

3.2

13

0

10

20

30

40

50

60

Medical ward Pediatricsward

OPD ICUs

Per

centa

ge

(%)

of

dia

rrhea

gen

ic E

. co

li

Sample origin

Chapter 4 Results

72

Table 7. Overall prevalence of diarrheagenic E. coli isolates

Variable Value Bacterial

Strains

Age distribution I month - 10 129 (18.43%)

11--20 184 (26.29%)

21-30 164 (23.43%)

31-40 77 (11%)

41-50 65 (9.29%)

51-60 42 (6%)

60-70 39 (5.7%)

Gender distribution Male 285

Female 430

Ward distribution Medical 91

ICU 37

Pediatrics 149

OPD 9

Among the 515 E. coli strains, 286 were isolated from the different wards of the

hospitals while remaining 229 E. coli strains were isolated from outdoor patients.

Chapter 4 Results

73

Annual Variation in Incidence of Diarrheagenic E. coli

Annual prevalence ratio was studied based on monthly sample collection and percentage

of E. coli isolation. In January, incidence rate was 0.52% (n=3), February 1.35% (n=7),

March 2.91% (n=15), April 4.66% (n= 24), May 16.89% (n=87), June 28.35% (n=146),

July 20.52% (n=106), August 13.01% (n=67), September 6.21% (n=32), October 3.3%

(n=17), November 1.75% (n=9) and in December it was 0.4% (n=2) (Fig 4.8).

Fig 4.8. Annual percentage isolation of diarrheagenic E. coli

0.52 1.35 2.91

4.66

16.89

28.35

20.52

13.01

6.21

3.3 1.75

0.4 0

5

10

15

20

25

30

Jan Feb Mach April May Jun Jul Aug Sep Oct Nov Dec

Per

centa

ge

of

DE

C i

sola

tio

n

Annual prevalence

Percentage Prevalence

Chapter 4 Results

74

Detection of E. coli O157:H7 among the Isolated Diarrheagenic E. coli pathotypes

During the present study, of 155 E. coli strains from age Group I, 22 (14.1%) were

identified as E. coli O157:H7. Similarly, number and percentage isolation of E. coli

O157:H7 strains from each respective age group is as follows: Group II: 17 (12.4%) of

137 strains, Group III: 8 (13.5%) of 59, Group IV: 5 (10.6%) of 47, Group V: 4 (9%) of

44, Group VI: 6 (14.2%) of 42 and Group VII: 2 (6.4%) of 31. However, none of the age

groups achieved statistical significance (Fig 4.9)

Fig 4.9. Percent detection of serotype O157:H7 among the isolated DAEC isolates.

22

17

8

5 4

6

2

0

5

10

15

20

25

Group I

(155)

II

(137)

III

(59)

IV

(47)

V

(44)

VI

(42)

VII

(31)

Pre

val

ence

of

E. co

li O

15

7:H

7

Age groups (I-VII) along with their no of DEC isolates (xx)

E. coli O157:H7

Chapter 4 Results

75

Seasonal Prevalence of Diarrheagenic E. coli

During the present study, 11.8% (61/515) strains were isolated during winter season

including 1.63% (1/61) E. coli O157:H7 serotype. During spring season 21.3%

(110/515), DEC strains were isolated including 7.2% (8/110) E. coli O157:H7 serotype.

Summer was found to be the peak season for DEC activities. During summer season

47% (242/515), DEC strains were isolated including 12.8% (31/242) E. coli O157:H7.

During autumn season 19.8% (102/515), DEC pathotypes were isolated, including 9.8%

(10/102) O157:H7 serotype as shown in (Fig 4.10)

Fig 4.10. Seasonal prevalence of DEC pathogroups

24

0

37

5

98

11

40

4

36

1

65

3

113

20

52

6

0

20

40

60

80

100

120

E. coli O157 E. coli O157 E. coli O157 E. coli O157

Winter Spring Summer Autumn

Gen

der

wis

e no

of

DE

C i

sola

tes

Seasonal specific DEC prevalence

Male

Female

Chapter 4 Results

76

Detection of Diarrheagenic E. coli in Water Sources

About 28% of pond waters (14/50) were contaminated with E. coli, of which 14% (2/14)

were identified as serotype O157:H7. Tap water was found to be free of serotype

O157:H7 contamination. Serotype O157:H7 was frequently isolated from sewage water

[16.13% (5/31)] and 38% (19/50) irrigation water samples were contaminated with E.

coli pathotypes, where (13.8%) 3/19 were identified as serotype O157:H7 (Fig 4.11).

Fig 4.11. Prevalence of DEC pathogroups from different water sources

14

8

19

31

2 0

3 5

0

5

10

15

20

25

30

35

Pond Water Tap Water Irrigation Water Sewage Water

Pre

val

ence

of

DE

C p

atho

typ

es

Water sources

No E. coli isolates

O157:H7

Chapter 4 Results

77

During the present study, 64 DEC pathogroups isolated from water sources were further

characterized for pathogroup specific virulence factors. 56.25% (36/64) of pathogroups

were identified as Enterotoxingenic E. coli, carrying a combination of elt/est virulence

gene. 28% (18/64) were identified as Enteropathogenic E. coli, consisting 72% (13/18)

typical enteropathogenic E. coli and 28% (6/18) atypical E. coli. Only 15.6% (10/64)

were identified as E. coli O157:H7 serotype carrying stx1 and stx2 genes (Fig 4.12).

Fig 4.12. Prevalence of ETEC, EPEC and EHEC in water sources.

6

2

3

1

2

10

7

6

3

5

7

4 4

1

3

0

2

4

6

8

10

12

LT ST tEPEC aEPEC stx1/stx2

ETEC EPEC STEC

No

of

DE

C p

atho

gro

up

s is

ola

ted

DEC pathogroups

Pond water

sewage water

Running water

Chapter 4 Results

78

Prevalence of DEC in Meat sources

About 58% (29/50) of beef meat sources were contaminated by E. coli, where 20.9%

(6/29) were identified as serotype O157:H7. Chicken meat samples were found free of

serotype O157:H7 contamination where E. coli was detected in 16% (8/50) samples.

Mutton samples were positively contaminated with serotype O157:H7 [21.5% (3/14)].

Approximately, 34% (17/50) goat samples were contaminated with E. coli, where only

one 1 among 17 (5.9%) was confirmed as serotype O157:H7 (Fig 4.13).

Fig 4.13. Prevalence of diarrheagenic E. coli pathotypes in meat sources

8

29

14

17

0

6

3 1

0

5

10

15

20

25

30

35

Chiken Beef Mutton Goat

No

of

DE

C p

atho

typ

e is

ola

ted

Meat soures

No E. coli isolates

O157:H7

Chapter 4 Results

79

Prevalence of DEC in Vegetables

About 54% (27/50) of salad samples were found contaminated with E. coli, where 14.8%

(4/27) E. coli isolates were identified as serotype O157:H7. (1) Cucumber was found to

be highly contaminated by E. coli 46%, (23/50), among some isolates 30.4% (7/23) were

confirmed as serotype O157:H7. (2) Spinach was also contaminated with E. coli by 28%

(14/50) where 28.57% (4/14) were identified as serotype O157:H7. Approximately, 40%

(20/50) of the (3) lettuce samples were contaminated by E. coli, where 15% (3/20) were

declared as E. coli O157:H7 (Fig 4.14).

Fig 4.14. Prevalence of diarrheagenic E. coli pathotypes in vegetable sources

27

23

20

14

4

7

3 4

0

5

10

15

20

25

30

Salad Cucumber Lettuce Spinach

No

of

DE

C p

atho

typ

es i

sola

ted

Vegetable sources

No DEC isolates

O157:H7

Chapter 4 Results

80

During the present study, 33 DEC strains were randomly selected from vegetable sources

for the detection of pathogroup specific virulence factors. 51.5% (17/33 of strains were

identified as Enteropathogenic E. coli, carrying a combination of lt/st virulence gene.

36.3% (12/31) were identified as Enteropathogenic E. coli, showed a presence or absence

of bfpA genes along with eae . E. coli O157:H7 serotype carried a combination of stx1

and stx2 genes (Fig 4.15).

Fig 4.15. Prevalence of ETEC, EPEC and EHEC in vegetable sources

5

2

6

4

3

5

3

1

0

1 1

2

0

1

2

3

4

5

6

7

Salad Lettuce Spinach Cucumber

No

of

DE

C p

atho

typ

es

Vegetable sources

ETEC

EPEC

EHEC

Chapter 4 Results

81

Antibiotic Susceptibility

Disc Diffusion Test

Diarrheagenic E. coli

Out of 300 DEC isolates, randomly selected for antibiogram development, included 150

(50%) E. coli from outpatients, 75 (25%) from medical ward and 75 (25%) from

pediatrics ward. 94% isolates were found sensitive to imipenem; cefuroxime was the

second most effective antibiotic (55%). The maximum resistance (92%) was observed

against tetracycline, followed by ampicillin 83% and ciprofloxacin 81%. In case of β-

lactam antibiotics, high resistance (78%) was observed against amoxicillin/clavulanic

acid (Fig 4.16).

.

Fig 4.16. Antimicrobial resistance patterns of E. coli O157:H7 serotype isolates.

83 81

61

56

51

45

56 52

92

77 81

78

6

17 19

39

44

49

55

44 48

12

33

19 22

94

0

10

20

30

40

50

60

70

80

90

100

Per

centa

ge

of

iso

late

s

Antibiotics

Resistant

Sensitive

Chapter 4 Results

82

Molecular Characterization

A total of 25 E. coli O157:H7 isolates were examined for the presence of shiga toxin

(stx) genes, a cardinal characteristic of EHEC group; stx1, stx2 and stx2c. They were also

sought for the locus of enterocyte effacement using LEE specific primers for tir, hly and

eae genes. Lineage assignment to the isolates was done through lineage specific

detection using primers with specificity for smfA, rbsB, Arp-icIR, rtcB, Z5935 and yhcG.

PCR amplification revealed that shiga toxin-producing gene was present among all

isolates. About 100% isolates were positive for the presence stx1 and stx2 genes. Stx2

variant stx2c was detected in 47% of the isolates. Presence of tir, hly and eae were found

to be the most common feature of EHEC serotype O157:H7, as 100% of the isolates

were positive for the presence of these genes.

Lineage I was found to be most dominant lineage (57%) among the tested isolates, 39%

belonged to Lineage I/II and 5% of the isolates belonged to Lineage II. Normal

housekeeping gene alignment showed 86% of the isolates kept an intact housekeeping,

while 13% isolates were showing deviation from normal formula, indicating novel

insertion deletion (Fig. 4.17, 4.18 and 4.19).

Fig 4.17. Ethidium bromide stained agarose gel showing PCR fragments for stx1, stx2

and stx2c gene of shiga toxin producing E. coli, Lane M: 1kb DNA ladder.

180 255

1000 900 800 700 600 500 400 300 200 100

180 255 255 255

180

Chapter 4 Results

83

Fig 4.18. Ethidium bromide stained agarose gel showing PCR fragments for eae, hlyA

and tir gene of shiga toxin producing E. coli, Lane M: 1kb DNA ladder.

Fig 4.19. Ethidium bromide stained agarose gel showing PCR fragments for smfA, rbsB,

Arp-icIR, rtcB, z5335 and yhcG of diarrheagenic E. coli pathotypes, Lane M: 1kb DNA

ladder.

1000 900 800 700 600 500 400 300 200 100

206

88 138

170

1000

900

800

700

600

500

400

300

200

100

218 324 270

472

133

Chapter 4 Results

84

Detection of Shiga Toxin Producing Phage Insertion Sites (SBI) in PA 4 and PA 33

Molecular characterization of E. coli O157:H7 was carried out for the Stx-encoding

bacteriophage insertion genotypes. Clusters 1 to 3 was observed 72% (18/25) of the

strains. 8% (n= 2) isolates were having of stx1, an occupied yehV site, and a shiga toxin

producing bacteriophage-occupied wrbA site that belongs to genotype 4. Additionaly

4% (n=1) isolate was characterized by the presence of stx2, the absence of stx1,

amplification of the right but not the left yehV-bacteriophage junction (designated yehV-

Variant- L), and an intact wrbA site belongs to genotype 5 (Fig 4.20 and 4.21).

Fig 4.20. Ethidium bromide stained agarose gel showing PCR fragments for stx1, stx2,

stx2c, yehv EDL 933,yehV left junction, wrbA EDL 933, wrbA right junction, sbsB and

argW of diarrheagenic E. coli pathotypes, Lane M: 1kb DNA ladder

180

700

600

500

400

300

200

100

124

282

702

289

1804

212

Chapter 4 Results

85

Fig 4.21. Ethidium bromide stained agarose gel showing PCR fragments for stx1, stx2,

stx2c, yehvV EDL 933,yehV left junction, wrbA EDL 933, wrbA right junction, sbsB and

argW of diarrheagenic E. coli pathotypes, Lane M: 1kb DNA ladder

Chapter 5 Discussion

86

Discussion

Diarrheal infections due to pathogenic E. coli pathotypes are one of the major public

health concerns around the world. An estimated mortality rate of diarrheal infections

exceeds well above 10 million in the developing world, rating it third in ranking of fatal

infectious diseases. Diarrheagenic E. coli pathotypes (DEPs) are well known diarrheal

infectious agents in Pakistan, causing a variety of infections, ranging from mild watery to

severe bloody diarrhea (Nguyen et al., 2005). Based on the stool physiology and

discharge periods, diarrheal infections are classified into three categories, namely; acute

watery, persistent, and bloody diarrhea. Former two forms of diarrhea (acute and

persistent diarrhea) represent two ends of a continuum, as they are not distinct from each

other, and showed almost identical number of stools discharged and appearance. Periodic

episodes of acute diarrhea resolve within the defined disease length (seven-day period)

but sometimes it may prolong beyond three to four weeks (McAuliffe et al., 1986).

Acute watery diarrhea, mostly occur during childhood in all population. World Health

Organization (WHO) defined “persistent diarrhea lasting for 14 days or slightly longer”.

Fourteen-day period is an arbitrary disease length, such lengthy unformed stool

discharge is enough for being fatal in children under the age of 5 years (Alam and

Ashraf, 2003). Bloody diarrhea contains visible or microscopic blood cells in stools, due

to local evasion of mucosal epithelial lining or may be due to intestinal hemorrhage

(Keusch et al., 2006). Periodically persistent diarrheal episodes are experienced South

Asia, in 10% diarrheal cases, with an estimated mortality rate of 35% to 50% (Bhan et

al., 1989).

Among the list of infectious agents, diarrheagenic E. coli pathotypes are regarded as an

emerging pathogen in resource-limited countries with an annual prevalence rate of 1.4

billion episodes among children less than 5 years of age (Parashar et al., 2003). Of 1.4


87

billion diarrheal infections 123.6 million episodes are severe enough that seek medical

consultation or outpatient medical care, whereas, 9 million episodes need ultimate

hospitalization (Khan et al., 1993). DEPs infections are expressed either with or without

associated symptoms i.e. vomiting, and abdominal cramps. Repeated episodes of

persistent diarrhea in infants impart cognitive effects and growth impairment that can

directly affect mental level and physical development (Black, 1991; Petri et al., 2008).

DEC associated diarrheal illness are more often related to growth impairment than any

other infections, that is why diarrheagenic E. coli infections is regarded as more

devastating than rotavirus infections (Mondal et al., 2009). Diverse nature of

diarrheagenic E. coli in respect of pathogenicity and antigens means that children and

adults may be challenged by the repeated diarrheal infection. So far, few studies on

diarrheal pathogens have been carried out in Pakistan (Bokhari et al., 2013).

Geographically North West frontier region of Khyber Pakhtunkhawa could be divided

into the northern and southern zone, stretching from the Hindu Kush mountainous ranges

to Derajat basin. The northern zone observes snow and more rains compared to southern

zone, whereas summer is hot and humid in both.

Southern areas of Khyber Pakhtunkhawa, Pakistan faced a sudden increase in diarrheal

cases during the floods in 2010 and 2011. This study showed that this epidemic was

largely attributed to different diarrheagenic E. coli pathotypes. During the present study

higher morbidity rate of diarrheal infection was observed among children during the

summer time, where abdominal pain and vomiting was observed as a trade mark

symptoms of DEC infection. Disease burden of diarrheal illness, prevalence data, and

molecular epidemiology of diarrheagenic E. coli pathotypes is rarely studied in Pakistan.

Darth of epidemiological data indicates lack of diagnostics facilities for the isolation and

identification of diarrheagenic E. coli pathotypes. Although it is unnecessary for testing


88

every diarrheal specimen for diarrheagenic E. coli pathotypes. Microbiological

examination of stool culture is expensive but on the other hand it could efficiently

decrease the treatment cost (Thielman and Guerrant, 2004). It is more suitable if disease

outbreaks of bloody, persistent, hospital acquired diarrhea are investigated for

diarrheagenic E. coli at sites of infection. Epidemiological surveys must be conducted to

devise risk management plans, treatment policy and development of new vaccine (Okeke

et al., 2000).

The purpose of this study was to assess the prevalence and molecular characterization of

diarrheagenic E. coli pathotypes, including (EPEC, ETEC and E. coli O157:H7) causing

diarrheal infections in southern parts of KPK, Pakistan, as well as their antimicrobial

resistance patterns. The present study is the first report in KPK on assessment of

antimicrobial resistance related to surveillance of E. coli pathotypes. Total 515 E. coli

pathotypes were collected from diarrheal stool specimens. Out of these, 64 isolates were

identified as E. coli O157:H7 using multiplex PCR technique.

Age Wise Prevalence of Diarrheagenic E. coli

Diarrheal infection is common in developing countries like Pakistan, where almost 100%

population experience a series of diarrheal episodes during puberty. By the age of 1 year,

nearly 90% of children have had at least one episode of infectious diarrhea. More than

90% of children with diarrhea are cared at home, less than 10% seek medical treatment,

and less than 1% gets hospitalized. Variations in the prevalence of diarrheagenic E. coli

are due to the variance in geographic position, as well with socioeconomic values.

During the present study, 57.22% (515/900) prevalence rate of DEC was observed.

Lower prevalence 2.2% of diarrheagenic E. coli was reported in Vietnam (Trabulsi et al.,

2002; Albert et al., 2009; Jafari et al., 2009). There are few comparable studies


89

concerning adult patients with diarrhea, while considerably more studies have been

conducted regarding childhood diarrhea (Caprioli et al., 1996; Presterl et al., 1999).

During the present study, 57% (292/515) of diarrheagenic E. coli were isolated from

patients of younger age (1-5 years). Comparatively, lower prevalence rate of 41.8% of

diarrheagenic E. coli among children was reported from Mozambique (Rappelli et al.,

2005). Similarly, 42.7% prevalence rate of DEC was reported by (Jafari et al., 2005) in

Iran among children less than 1-year-old (Jafari et al., 2009). Comparatively low

incidence rate of 10% was reported in children from Djibouti (Mody et al., 2007; Dutta

et al., 2011) and 17% in Bangladeshi children (Mody et al., 2007). 34% prevalence rate

of diarrheagenic E. coli in children less than 5 years old (Brooks et al., 2006). Earlier

studies in Pakistan have documented the prevalence of some traditionally recognized

diarrheal agents instead of diarrhaegenic E. coli detection. Higher (63.3%) incidence of

diarrheagenic E. coli was reported by Farid Abu-Elamreen in children in their first year

of life (Abu-Elamreen et al., 2008). Children less than 5 years of age experienced

approximately 3.2 diarrheal episodes with maximum of 4.8 episodes during the first year

of life. With the growing age, immune system is developed and thereby progressively

declined the rate of diarrheal episode to 1.4 episodes per year to the age of 5 years.

Infantile diarrhea is more alarming during 1st year of life exhibiting high mortality of (8.5

children per 1000/year) (Kosek et al., 2003). During the present 30% E. coli, isolates

were isolated from age I (from 1 month up to 10 years). Whereas, 26.6% (137/515) E.

coli strains were isolated from age group II (11-20 years). Similarly, 11.5% from age

group III (21-30 years), group IV (9.2%) group V (8.5%), group VI (8.2%) and group

VII (6%). These results are in accordance with Jordanian study (Shehabi et al., 2003)

showed that higher proportion of diarrheagenic E. coli is obtained from younger

individuals compared to adults. DEC prevalence of such a higher ratio among children


90

could be due to effect of environmental exposure and increased ingestion of solid foods

to children whose immune system is still in the developmental stages.

Seasonal Prevalence:

Diarrheagenic E. coli showed a higher morbidity rate during summer season. It is also

evident that DEC can transmit longer during rainy season, suggesting water as a possible

route of transmission. In the present study 47.6% (245/515) DEC pathotypes were

isolated during the summer season (May-Aug 2011). These reports are in accordance to

the study from Tanzanian where 34.6% prevalence of diarrhaegenic E. coli was reported

in the dry season and in the rainy season to over 28-70% overall (Vargas et al., 2004). A

summer peak of pediatric diarrheal infection was recorded in Taiwan. Similarly, high

trend in the prevalence of diarrheagenic E. coli during summer (Jafari et al., 2009) as

42.7% in Iran among children less than 1 year of age. Higher detection rate during the

summer season may be explained due to high temperature during this time of the year is

thought to promote growth of these organisms in the environment. Moreover, monsoon

season help to emerge pathogenic strains from inadequate sewage disposal system, and

also brought to them to the food chain through water cycle, therefore resulting in a

greater number of incidents. Occurrence of diarrheal infection is inclined during hot and

humid summer season. Emergence of enteric pathogens in food items is favored by hot

and humid summer, explaining higher prevalence of diarrheal infection during summer

(Wright, 1986; Lee and Middleton, 2003). Heavy rainfall causing flood are responsible

for almost all waterborne E. coli outbreaks. E. coli flourished in hot and humid summer

season (July, August and September). During the present study 47% (42/90) of E. coli

pathogroups were isolated during summer season (May-August). About 8% (7/90), E.

coli strains were also obtained during the winter season (October-January).


91

Transmission and season specific prevalence of E. coli pathogroups are attributed to the

increased housefly populations in the summer (Alam and Zurek, 2004), environmental

factors (humidity and rainfall) (Gagliardi and Karns, 2000) and waste handling practices

(Rasmussen and Casey, 2001; Perencevich et al., 2008). Spring and autumn were

observed as the less effective seasons for E. coli pathogroup activities 20% (18/90) and

9% (23/90) respectively. It is evident that E. coli pathogroup survival is favored by the

environmental factors, like temperature and heavy rainfall.

Prevalence among Gender:

During the present study, 60% DEC isolates were obtained from female as compared to

40% male. A study in Palestine showed higher incidence of diarrhea in females 59% as

compared to 41% male (Abu-Elamreen et al., 2008). These results are in conformity with

those obtained by (Beutin et al., 1998) showed 54% prevalence of diarrheagenic E. coli

among female. Contrary to the present study (Jain et al., 2003) has reported 61.4%

incidence rate of diarrheal infection among males as compared to 38.6% in females.

Similarly, 61% prevalence rate among male was reported by (Dutta et al., 2000).

Diarrheal morbidity/mortality rate among children less than 1 year is on the rise in

Pakistan, as proven by the present study; incidence rate of diarrheal infections among

female child is 70% compared to 66% male patients. E. coli strains were isolated from 58

% children less than one-year-old (Abu-Elamreen et al., 2008). Contradictory, to the

present study 73% DEC prevalence among male child (Taneja et al., 2011). Higher

prevalence 61% of DEC pathogen among male children is also reported (Dutta et al.,

2011).

Based on the origin of specimens in our study, 50% (260/515) isolates were from

different wards of the hospitals consisting of 11.15% (29/260) from medical ward, 26.9%


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(70/260) from OPD, while 54% (141/260) were isolated from Pediatric ward, and only

7% (20/260) were isolated from different ICUs. In Tanzania isolated 22.9% (64) E. coli

strains from 280 diarrheal children under five years of age (Moyo et al., 2007). 53.8%

prevalence of E. coli in diarrheal children and 53.1% prevalence in control group were

observed in Nicaragua (Vilchez et al., 2009). During 1940, 10% to 20% of children with

diarrhea required hospital admission, with a mortality rate of 5-45% in Taiwan.

Prevalence in Vegetables

E. coli species are most frequently used as an indicator organism for maintenance of food

hygienic quality. Food items are contaminated during processing either through usage of

raw materials, through carriers‟ food handlers, or may be due to the poor hygiene

condition of the store or during the transportation. High microbial contamination of the

leafy vegetables, carrots and sprouts was reported (Samadpour et al., 1994) for

minimally processed fresh vegetables. Generally, sprouts, grated carrot, arugula and

spinach, with lettuce (different cultivars) are observed with high microbial load. Carrots

being subterranean crop are directly exposed to contaminants from soil, water, manure,

fertilizers and irrigation water. Spinach is topsoil crop, but due to open leaves is exposed

to the contaminants from soil and irrigation water. Sprouts have been identified at higher

risk for potential pathogen growth during the sprouting period. Consumption of

contaminated food item whether of animal or plant origin could leads to either foodborne

outbreaks or sporadic cases of diarrheal infections due to E. coli strains. The importance

of detecting diarrheagenic E. coli pathotypes in fresh vegetables was highlighted by a

foodborne outbreak of hemolytic uremic syndrome and bloody diarrhea due to E. coli

O104:H4 associated with sprout consumption in Germany (Buchholz et al., 2011; Gault,

et al., 2011).


93

In the present study, 54% (27/50) of salad samples were contaminated with E. coli, and

14% (4/27) of them were identified as E. coli O157:H7. These results are potentially

lower than a reported 20% isolation in the United Arab Emirates (UAE) (Almualla et al.,

2010) and 16.7% in Spain (Abadias et al., 2008). Comparatively lower incidence rate as

1.3% of E. coli O157:H7 was reported in the United Kingdom (Sagoo et al., 2003).

Contrary, to the present study, higher prevalence of 89% and 73% of fecal coliform (FC)

were reported from Costa Rica (Rodriguez-Cavallini et al., 2010) and from Brazil

(Froder et al., 2007), respectively. During the present study, 40% of lettuce samples were

contaminated with E. coli pathotypes, where 15% (3/20) were identified as E. coli

O157:H7 serotype. Cucumbers are often regarded as potential carrier of E. coli O157:H7.

In the present study 46%, (23/50) cucumber samples were contaminated with E. coli

strains, and 30% of these strains were identified as serotype O157:H7. Spinach is a

source of E. coli foodborne outbreaks. In the present study, 28% (14/50) spinach samples

were contaminated with E. coli pathotypes, and 28.6% (4/14) were identified as E. coli

O157 serotype. These results are in agreement with the previous studies in Netherland

reported 10.4% (Cetinkaya et al., 2000) and 13.4% in England (Chapman et al., 1997).

Comparatively lower prevalence rate of 2% in sprout samples is reported from Norway

(Robertson et al., 2002). High prevalence of about 86.1% and 75% of vegetables

contamination was reported from Germany in 1987 (Garcia-Villanova et al., 1987).

None of the vegetables and fruits sample was contaminate in a survey carried out in

Hong Kong (FEHD, 2002), USA (Mukherjee et al., 2006), UK (Sagoo et al., 2003),

Ireland (McMahon and Wilson, 2001), and Norway (Johannessen et al., 2002).

Moreover, E. coli pathotypes were responsible for the recent foodborne disease

outbreaks due to the consumption of fresh fruits and vegetables (Soderstrom et al.,

2005). In the present study, higher prevalence of DEPs could be due to the fact, that


94

agricultural farms are favorably irrigated with untreated sewage water due to its manure

contents. Thus, contamination is most likely due to the irrigation water along with poor

sanitation practices at the nearby area. Main reason is that most of the root vegetables

(carrots, onions and radish) and leafy green vegetables (lettuce, spinach, coriander) are

irrigated with sewage water. About 16% (8/50), salad samples having DEP strains were

identified, such as 62.5%, ETEC and 37.5% were EPEC. Present study is an evident for

the isolation and characterization of DEPs from ready to eat (RTE) salads in Pakistan.

Similarly, EPEC and ETEC strains were frequently from local restaurants and beverage

in Mexico city (Lopez-Saucedo et al., 2010; Cerna-Cortes et al., 2012). Diarrheagenic E.

coli is frequently from foodborne disease outbreaks due to ingestion of fresh products

e.g. E. coli O104:H4 (Buchholz et al., 2011).

Unfortunately, agricultural land of Southern Khyber Pakhtunkhawa, Pakistan is irrigated

either with wastewater and stored rainy water since no time and will remain continue for

centuries because of no wastewater treatment facilities. To interrupt the transmission line

of enteric pathogens from cattle to water, and from water to the human through food,

Insurance for the irrigation of root and leafy vegetables farms with clean running water,

this is only option that could reduce the transmission load and thereby, help in the

reduction of series of GIT infections caused by these pathogens.

Prevalence in Meat sources

During the present study 16% (8/50) chicken meat samples were observed contaminated

with E. coli pathotypes, but none of these were identified as E. coli O157:H7 serotype.

Comparatively high prevalence of E. coli O157:H7 was reported (4.0%) in Korea (Jo et

al., 2004) and 10.3% in Egypt (Abdul-Raouf et al., 1996). Similarly, during the present

study, 58% (29/50) of the beef samples were contaminated with E. coli pathotypes and


95

28% (6/28) of them were identified as serotype O157:H7. Comparatively, high

prevalence rate of 74.5% of serotype O157:H7 was reported in South Africa as compared

to 36% in Malaysia (Radu et al., 1998). Prevalence of STEC is well documented among

various beef and cattle categories (Shinagawa et al., 2000; Schurman et al., 2000;

Gannon et al., 2002). STEC can be isolated from dairy farms (LeJeune and Christie,

2004) and under grazing conditions (Cobbold et al., 2004). Lower prevalence rate of

serotype O157:H7 in beef and cattle sample is reported from Argentina 0.17% (Chinen et

al., 2001), Egypt 1.4% (Abdul-Raouf et al., 1996), Switzerland (2.3%) and Ethiopia

(3.8%) (Hiko et al., 2008). Several studies have showed beef and its products free of E.

coli contamination (Uhitil et al., 2001) or yield lower contaminated sample rates (Itoh et

al., 1999). Higher prevalence of DEPs is evident that unhygienic conditions prevail in

slaughterhouses of Pakistan. STEC strains have been isolated from diverse range of

animals (Beutin et al., 1993; Beutin et al., 1995). Cattle serves as a primary host for the

diarrheagenic E. coli pathotypes, involved in a number of foodborne diarrheal infection

(Crump et al., 2002; Lahti et al., 2002). In the current study, 34% (17/50) of the goat

meat samples were contaminated with potential E. coli pathotypes, 5.9% (1/17) of the

isolates were identified as serotype O157:H7. These results are lower than reported in a

study in Iran, showed 11.6% prevalence (19/159) in sheep meat samples (Doyle and

Schoeni, 1987) and higher than 2.5% prevalence rate of E. coli O157:H7 in a study in

Ethiopia (Hiko et al., 2008). Low prevalence of 2.0% of E. coli O157:H7 in sheep and

goat meat samples (Momtaz and Jamshidi, 2013), (0.77%-7.3%) is in Italy (Duffy et al.,

2001), 4.0% in Egyptian, 1.5% in USA (Duffy et al., 2005) and 0.5% in Australia (Jo et

al., 2004).


96

Prevalence in Water Sources

Water bodies including rivers, ponds, and marshy places are serving as nursery house for

microbes. Water bodies are directly contaminated by the toilet run off water, industrial

effluents, defection in or near catchment areas and flooding (Roe et al., 2003; Lupo et

al., 2012). This problem is even more severe in the remote areas of Pakistan, where

untreated sewerage water and industrial effluents are directly released to the water

sources without prior treatment. Animals are mainly responsible for shedding E. coli

pathogroups into the environment, and remain asymptomatic by lacking

globotriaosylceramide (gb3) receptor. This contaminated water is subsequently used for

irrigation, without any prior treatment (Mazari-Hiriart et al., 2008; Jang et al., 2013). In

rural communities, this extensively contaminated water is not only used for irrigation

purpose but also for drinking and watering of livestock. Poor sanitation and unhygienic

conditions are a major source of water contamination. Agricultural practices that involve

usage of sewage waste water and/or water carrying cattle manure on farms are primarily

contributed in the completion of E. coli pathotypes life cycle from cattle to human beings

and vice versa. Similarly, uncontrolled waste disposal, bathing and swimming in water

sources such as rivers and dams, which are used as sources of municipal water supplies

are a major cause of water contamination. During the present study, 16% (8/50) of tube

well water were found contaminated with E. coli pathotypes, but none of these were

identified as E. coli O157 serotype. During the present study, 38% (19/50) running

stream water (used for irrigation) samples were contaminated with DEC and 5% of them

were identified as E. coli O157 serotype. During the present study, prevalence rate of E.

coli pathotypes is observed higher compared to 10.7% and 16.7% of E. coli

contamination in different wells. 58.33% prevalence of E. coli pathotypes was reported

in tube well water (Figueras and Borrego, 2010).


97

In Southern Khyber Pakhtunkhawa, Pakistan, drinking water sources both urban and

rural areas are being well managed. Almost all of the drinking-water reservoirs are fecal

contaminated due to surface run off or during cattle watering. Groundwater is also

contaminated due to subsoil contaminants or to sipping of the run-off water.

Bacteriological contamination of drinking water subsequently resulted in higher

incidence of waterborne diseases (Aziz, 2005).

Some restriction must be undertaken not to irrigate routinely used vegetables (carrot,

onion, radish lettuce, spinach and coriander) with untreated sewage water (Sagoo et al.,

2003). In the present study, about 62% (31/50) of sewage water samples were found

contaminated with E. coli pathotypes, and 16.2% (5/31) of them were identified as E.

coli O157:H7 serotype. Pond/stagnant water bodies were also found to be contaminated

with potential virulent strains of E. coli pathotypes. In our study, about 28% (14/50) of

stagnant water samples were contaminated with E. coli, and 14% (2/14) of them were

identified as E. coli O157:H7. This particular strain was found to be higher concentration

in stagnant water commonly used for domestic cattle watering. This finding is consistent

to a study showed high prevalence rate of E. coli O157: H7 in pond water sample (Hill et

al., 2011). In Pakistan like other developing countries, sewage and wastewater are

contributing more in the environmental pollution. Animal farming in such countries are

without adequate disposal system which in another sign of threat for the aquatic systems.

Contaminated running water after from these activities is subsequently used for

irrigation, without any treatment (Jang et al., 2013).

An efficient transmission of pathogens has been observed during flooding situation. It

may be due to mixing of fecal wastes of both animal and human origin present in the soil

and other sources to the water bodies. During the present study, higher number of E. coli

pathotypes 59% (53/90) was obtained after heavy rainfall resulting in a flooding situation


98

in southern areas of KPK, Pakistan. A potential cause could be the addition of human

and animal excreta from sewerage leakage and overflow (Davies and Bavor, 2000),

transmission of E. coli present in the soil (Brennan et al., 2010), sediments (Lekowska-

Kochaniak et al., 2002) and water (Lothigius et al., 2010). These findings are in

accordance with the previously reported observation of several fold increase in the

number of E. coli pathotypes in the surface waters after storm (Brownell et al., 2007;

Parker et al., 2010). This highlights the importance of managing municipal wastewater

including sewerage leaks and overflows in aquatic environments.

Antibiotic Resistance:

Globally, antibiotic resistance of enteric pathogens has reached to alarming proportions.

Emerging antibiotic resistance is attributed to the misuse and self-prescription giving

microorganisms a chance of better survival (Okeke et al., 1999). In the last few decades,

antibiotics were excessively used in disease prevention and for attaining rapid animal

growth/ dairy products, thereby giving a chance to the resistant microbial phenotype to

modify accordingly. Human beings are directly exposed to the resistant pathogens

through food and drinking water contaminated with feces etc. During diarrheal onset,

antimicrobials should be precisely prescribed to shorten the disease period. Conversely,

antimicrobial treatment may decline disease burden by eliminating shedding of Vibrio

cholerae and E. coli strains.

E. coli pathotypes i.e. EPEC, ETEC, EIEC, and EAEC are 100% resistant to available

drugs to patients in Southern KPK, Pakistan, so that optimal treatments does not exist

(Adetosoye, 1980; Bii et al., 2005). Studies have reported that 25-90% of ETEC, EAEC

and EPEC isolates were resistant to ampicillin, trimethoprim-sulphamethoxazole,

tetracycline and chloramphenicol. Resistance to the quinolones, although then


99

uncommon, was detected, which is highlighting the only option for the treatment of

pediatric persistent or invasive diarrhea in many areas (Vila et al.,1999). During the

present study, maximum resistance (98%) was observed against tetracycline. These

results are in accordance with the previous reports, showed that 91% tetracycline

resistance among E. coli is prevailing in Pakistan (Alam et al., 2003). About 90% of the

diarrheagenic E. coli showed resistance against tetracycline that intensifies the need to

screen these strains for tetracycline resistant genes (tetA to tetD). E. coli isolates 63% of

the humans and animal origin carried tetB gene and tetA (Bryan et al., 2004; Sengelov et

al., 2003). Higher ratio of tetracycline resistance observed during the present study might

be due to the presence of tetA genes that were not tested or it could be due to the

presence of integrons that are easily transferred through conjugation (Fischer et al.,

2001). In case of β -lactam antibiotics, 87.8% resistance was observed against

cefotaxime and amoxicillin/clavulanic acid, followed by cefepime (81.7%) and

aztreonam (79.4%). About 33.6% of the isolates were resistant to amikacin, while 80.9%

showed resistance to ciprofloxacin. Whereas, 100% isolates were found sensitive to

imipenem. Comparatively, low resistance of 51% and 60% is reported in uncomplicated

and complicated strains of E. coli (Yuksel et al., 2006). The ampicillin resistance among

urinary tract pathogens is probably due to its continuous use for many years. Earlier it

has been reported that ampicillin has no more effect on urinary tract pathogens (Sahm et

al., 2000). Tetracycline resistance is emerging in clinical isolates of our community. In

one study tetracycline resistance was found to be 83.9% showing consistency with the

present results (Noor et al., 2004). Resistance to ciprofloxacin was found to be 95%

among ESBL producers and 56% non-producers, which is high as compare to other

report from Pakistan (Alam et al., 2003). The susceptibility of ciprofloxacin is quite high

among ESBL producing E. coli isolates in Korea (Poppe et al., 2005). In Pakistan, this


100

resistance may be due to the use of flouroquinolones as a drug of choice in UTI

infections. Ceftazidime resistance was 38.3% against among ESBL producing isolates of

E. coli was reported (Poppe et al., 2005). Present study shows that cephalosporin

resistance is also on the rise in Pakistan, where resistance against ceftriaxone and

ceftazidime was observed as i.e. 54% and 52%, respectively. Results of ceftriaxone

resistance in present study are lower than 73% as reported in Iran (Mehrgan and Rahbar,

2008). There is evidence indicating that tetracycline and quinolone survives longer in the

environment than other antibiotics, which may be critical in maintaining the level of

resistance at a high. Antibiotic resistance of E. coli to tetracycline, ampicillin, and

ciprofloxacin observed during the present study are similar to those obtained (Okoli and

Iroegbu, 2004; Okeke et al., 2011).

Gentamicin resistance observed during the present study was 62%, which is high as

compared to previous study (Alam et al., 2003) indicating the uncontrolled usage of this

drug. These results are high enough as compared to the previous reports (Narchi and Al-

Hamdani, 2010). Generally, in the United States and Canada, E. coli isolates from

patients with UTI display >95% susceptibility to fluoroquinolone (Barnett and Stephens

1997).

Imipenem is a carbapenem antibiotic, which is highly active against enterobacteriaceae

producing ESBL. This drug is highly β -lactamase stable and has an unusual property of

causing a post antibiotic effect on Gram-negative bacteria (Paterson and Bonomo, 2005).

During the present study, 87% of DEC pathotypes were sensitive to imipenem. Similar

results were observed in another study that imipenem demonstrated excellent activity

against a wide variety of Gram positive and Gram-negative bacteria, including ESBL-

producing organisms (Franiczek et al., 2005; Franiczek, et al., 2012). For this reason,

tigecycline could be considered as an encouraging antimicrobial for the treatment of


101

infectious diseases. However, this antibiotic is not recommended in adolescents under 18

years of age due to lack of data on its safety, and should not be used in children under 8

years of age because of tooth discoloration.

E. coli isolates from chicken, showing resistance to tetracycline (99.1%), spectomycin

(95.7%), Trimethoprim-sulfamethazole (92.2%), gentamicin (89.7%), ampicillin (88.7%)

and Chloramphenicol (57.0%) (Al-Ghamdi et al., 1999).

Consequences of antibiotic misuse resulted in generating antimicrobial resistant bacteria.

Improper use or inappropriate choice of antibiotics exerts selective pressures on

dissemination of resistant genes in pathogenic bacteria in clinical environments (Tacao et

al., 2012). During the present study, E. coli pathogroups isolated from sewerage water

showed highest antibiotic resistance against tetracycline and ciprofloxacin. This suggests

the importance of wastewater discharges in the dissemination of antimicrobial resistant

strains. About two thirds of all E. coli isolates were found resistant to ampicillin,

trimethoprim-sulfamethoxazole and chloramphenicol. The presence of antibiotic

resistant E. coli was also observed in other studies from human and animal fecal sources,

wastewater treatment plant and surface water (Sayah et al., 2005; Ibekwe et al., 2011;

Mokracka et al., 2011). Similar resistance levels were found in E. coli isolated from

children and adults in Latin America, where 53.2 and 57.7% isolates were reported

resistant to ampicillin and trimethoprim-sulfamethoxazole, respectively (Estrada-Garcia

et al., 2005). Our results revealed the presence of pathogenic E. coli in the running water,

with multidrug resistance characteristic, including β-lactam resistance, an antibiotic

highly used in humans and animals. This situation highlights the risk of dissemination of

multidrug resistant pathogens (Dalhoff, 2012). The permanent influx of pollutants such

as antimicrobial agents, detergents, disinfectants, heavy metals, livestock waste and


102

watershed have contributed to the emergence of antibiotic resistant bacteria in water in

Southern Khyber Pakhtunkhawa, Pakistan.

Molecular Characterization

Molecular risk assessment played a crucial role in grouping of diarrheagenic E. coli

pathotypes. Among others, serotype O157 causes life-threatening outbreaks in humans.

Non-LEE effector genes determined by O-islands OI-122, OI-71 and OI-57 are mainly

responsible for food based human outbreaks (Bugarel et al., 2010). The advent of O157

and non-O157 EHEC strains in epidemic human disease outbreaks is of global concern

(Coombes et al., 2008). During the present study, 7.8% (40/515) of isolates were

identified as shiga toxin producing E. coli (STEC) strains based on the presence of Stx

genes. These results are lower than that reported 9% in South Africa and 10% in

Tanzania (Rajii et al., 2008). Comparatively, higher prevalence of STEC was reported

35.5, 25.5, 21.7, 56.5% and 43.5% among meat products, water, vegetables, and stools in

India (Abong'o and Momba, 2008).

E. coli O157:H7 strains isolated during the present study were positive for shiga toxin

producing genes (Stx1, or Stx2), tir, eae, and hlyA genes. Similar observations were

reported in the previous studies, showed 96% prevalence of the eaeA gene in E. coli

O157:H7 isolates from water (Shelton et al., 2006; Masters et al., 2011). Presence of eae

gene express for intimin which aid in binding to host cell (Oswald et al., 2000) and act as

an accessory gene for the disease onset (Gyles et al., 1998; Paton and Paton, 1998;

Beutin et al., 2005). Presences of hlyA gene among all clinical isolates of the E. coli

O157 isolates show its easy exchange in E. coli strains (Schmidt et al., 1994). During the

present study 100%, isolates were positive for hlyA genes. Similarly, 100% presence of

hlyA is reported (Tarr et al., 1999). Presence of 100% hlyA gene may suggest its role in


103

colonization (Frydendahl, 2002). Bloody diarrheal infections caused by EHEC

pathotypes may leads to HC or HUS (Boerlin et al., 1999). eaeA gene is mostly found in

combination with Stx1 genes (Paton and Paton, 1998).

During the present study 100% EPEC, categorized as either tEPEC or aEPEC isolates

were carrying eaeA gene alone or in combination of bfpA. As previously reported in

developing countries, gastroenteritis is mainly due to the atypical EPEC pathotype that

carries eaeA gene but lacking the bfp gene (Hernandes et al., 2009; Robins-Browne et

al., 2004; Kozub-Witkowski et al., 2008). A combinatorial pathogenicity mechanism is

adopted by all E. coli pathogroups, consisting of attachment and effacement,

modification of host‟s cell surface, invasion of the brush border epithelial cells, secretion

of toxins (Boerlin et al., 1999). Mostly tEPEC strains were carrying the LEE encoded

PAIs containing intimin (eaeA) and the plasmid-borne bundle forming pilus (bfpA),

which helps in binding to the intestinal epithelial cells (Kaper et al., 2004; Hamilton et

al., 2010). Identification of virulence genes is often helpful in determining pathogenic

properties of a given E. coli pathotypes (Kuhnert et al., 2000). Likewise, high prevalence

of the eaeA gene in surface water has been reported in other studies (Masters et al.,

2011). Furthermore, our study was partially conducted in densely populated flood

refugee camps, a setting that is often encountered in developing countries and that must

be taken into account (Mazari-Hiriart et al., 2008). This poses a potential risk for human

infections because water is used for consumption or for recreation (Hamelin et al., 2007).

These results showed that there is an urgent need to evaluate the management of

wastewater and the water quality in Khyber Pakhtunkhawa and if necessary, install local

wastewater treatment plants to prevent the emergence of infectious diseases outbreaks.

Our results revealed the presence of pathogenic E. coli in the food items with multidrug

resistance characteristic, including β-lactam resistance, an antibiotic highly used in


104

humans and animals worldwide (Dalhoff, 2012). The permanent influx of pollutants such

as antimicrobial agents, detergents, disinfectants, heavy metals, livestock waste, and

watershed may contribute to the emergence of antibiotic resistant bacteria in water as

well as the spread of antimicrobial resistance genes in southern Khyber Pakhtunkhawa.

E. coli O157: H7 while being primarily associated with food-borne outbreaks has also

become an important public health concern as a water-borne pathogen. This pathogen is

getting more dominance in developing countries, due to lack of diagnostic and treatment

facilities.

Pathogenicity of E. coli O157:H7 is attributed to the secretion of shiga toxin and binding

to the intestinal epithelial cell using LEE-encoded proteins. Sequential gain of Stx

encoding phages is the key process in the evolution of this pathogenic clade from

commensal E. coli. Other pathogenic E. coli belonging to this clade differ in the level of

Stx production or LEE encoded effector proteins or they may contain other unrecognized

virulence genes. Phage movements are consistent with the occupancy of wrbA site and

the presence of Stx2c gene accompanied by the additional insertions of other phages. It is

proposed that sequential insertion of Stx-encoding bacteriophages in specific

chromosomal locations of E. coli O157:H7 (Shaikh and Tarr, 2003).

Strategies for Controlling Antibiotic Resistance

This high degree of resistance could be explained by the fact that antibiotics are readily

available without physician‟s prescription from almost all the medical stores. Self-

prescription and usage of cheaper antibiotics for all sorts of infections by patients,

quakes, doctors and dispensers, and taken in inadequate doses resulting in high degree of

resistance. Given that majority of chemotherapy available for UTI is inadequate where

UTI pathogens are often exhibiting increasing antibiotic resistance. Usually restriction of


105

one class/generation of antibiotics can lead to increased use of another class with an

accompanying increase in resistance rates. Various attempts have been attributed to

decrease the prevalence of ESBL producing UTI pathogens by substituting older version

of cephalosporin with a fourth-generation cephalosporin or β-lactam/β-lactamase

inhibitor combinations (Taneja et al., 2011).

This study highlights the need for an antibiotic policy for its rationale use in the country.

The policy should stress not only on prevention of infections, but also ensures proper

selection of antibiotics and there should be minimum misuse of antibiotics. Clinicians

must depend on more laboratory guidance, while laboratories must provide resistance

pattern data for optimal patient management more rapidly. Our findings provide some

necessary information about the existence of E. coli O157:H7 in cattle‟s meat, vegetables

and drinking water that could be used in future studies. Finally, there is also a need to

improve on infection control methods.

Present study has certain limitations such as that no control group was tested for the

identification of specific risk factors or to compare the prognostic suggestions. Although,

our screening protocol was designed to study the prevalence of diarrheagenic E. coli

pathotypes, which might not be founded in some cases. Finally, low number of cases in

some subgroups preclude from obtaining robust conclusions.

In conclusion, it is highly recommended, that accumulation of wastewater into the

drinking or irrigation water must be eliminated or reduced, in order to decline diarrheal

disease burden. This study also highlights installation of local wastewater treatment plant

for the environmental safety and breaking of infectious diseases transmission chain.

Present study highlights the immense need of developing a better understanding of

public health implications for the prevalence of E. coli pathotypes in water and food

sources.


106

Conclusions

106

Conclusions

Diarrheagenic E. coli pathotypes, emerging foodborne pathogens posing serious

issues of acquired infections.

Diarrheagenic E. coli pathotypes accounted severe infections as compared to

commensal E. coli spp in the positive samples.

Differential media were successful for the detection of diarrheagenic E. coli

pathotypes in mixed pathogens.

Enterotoxingenic E. coli pathotypes showed higher prevalence compared to

Enteropathogenic E. coli and other pathogroups.

Out of these 515 isolates, 300 samples of the phenotypically E. coli were

processed for antibiotic resistance. Imipenem was the most effective antibiotics.

Maximum resistance was observed against tetracycline, ciprofloxacin and

sulphamethoxazole. Higher resistance was observed for all six β-lactam

antibiotics tested.

Higher prevalence of diarrheagenic E. coli in age group I showed, that Age was

significant risk factor for diarrheagenic E. coli pathotypes.

Stx1 and Stx2 was the most prevalent (96%) out of three-shiga toxin producing

genes tested (stx1, stx2, stx2c) in shiga toxin producing E. coli (STEC). This

trend is similar to that observed in other countries of Europe and Asia. Stx2c was

detected only 4% of STEC strains, indicating that these genes are newly

accumulated in Pakistan.

Presence of 100% (eae, tir and hlyA) in STEC isolates indicates persistent nature.

This trend of gene combinations was commonly found in the present study.

Conclusions

107

Shiga toxin producing bacteriophage insertion (SBI) analyses showed yehV and

argW are occupied by bacteriophages from both sides, commonly found in the

present study.

Future Prospects

108

Future Prospects

Prevalence of diarrheagenic E. coli pathotypes in foodborne outbreaks is rarely

studied prospective of medical research in Pakistan. There is an immense to

establish a database for foodborne diarrheal outbreaks.

Antibiotic resistance is on the rise in Pakistan, but unfortunately, it is the most

rarely investigated area of medical research in Pakistan. Monitoring database of

antibiotic resistance is immensely required for the analyses of antibiotic

resistance level and its association with various risk factors in Pakistan.

Self-medication, Low or over dosage of antibiotics is very common in Pakistan,

due to which most of the antibiotics are not working properly. For the

development of better risk management, extensive research in antibiotic

resistance is required.

Further studies are required to evaluate the of pathogroups specific prevalence of

diarrheagenic E. coli, impact of antibiotic selection, dynamic flow of organisms

between food and community, and the multiple origins of the isolates.

Pulsed Filled Gel Electrophoresis analysis could be performed to detect the

source of the infection.

Further studies are required to detect novel genes and novel mutations.

Chapter 6 References

109

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Appendix

A- 1 -

A-1. Overall distribution of diarrheagenic E. coli among the study group

Stool samples Watery Diarrhea Mucoid stool Bloody Diarrhea Total

DEC positive Number (n) 272 197 46 515

Percentage (%) 52.8 38.2 9 100

DEC negative

Number (n) 168 194 23 385

Percentage (%) 43.6 50.4 6 100

Total

Number (n) 440 391 69 900

Percentage (%) 49 43.3 7.7 100

A-2. Overall distribution of diarrheagenic E. coli among different age categories.

Bacterial Isolate Age Groups (Years)

Total

1 month-10 years

11--20

21-30

31-40

41-50

51-60

Above 60

E. coli Number (n) 133 120 51 42 40 36 29 451

Percentage (%)

29.5 26.6 11.3 9.3 8.9 8 6.4 100

E. coli O157:H7

Number (n) 22 17 8 5 4 6 2 64

Percentage (%)

34.4 26.6

12.5 7.8 6.25 9.4 3.05 100

Total

Number (n) 155 137 59 47 44 42 31 515

Percentage (%)

30.1 26.6

11.5 9.1 8.5 8.2 6 100

Appendix

A- 2 -

A-3. Percentage distribution of diarrheagenic E. coli on the basis of sample origin

Bacterial Isolate

Sample Origin

Total Medical ward Pediatrics ward OPD Medical ICU

E. coli Number (n) 91 149 9 37 286

Percentage (%) 31.8 52 3.2 13 100

A-4. Seasonal prevalence of DEC pathogroups

E. coli pathotypes Winter Spring Summer Autumn Total

E. coli

Male Number (n) 21 37 97 40 195

Percentage (%) 11 19 49 21 100

Female Number (n) 29 62 113 52 256

Percentage (%) 11 25 44 20 100

E. coli O157:H7

Male Number (n) 1 5 17 3 26

Percentage (%) 3.8 19.2 65.4 11.6 100

Female Number (n) 0 3 24 11 38

Percentage (%) 0 7.9 63.2 28.9 100

Total Number (n) 51 107 251 106 515

Percentage (%) 9.9 20.8 48.7 20.6 100

Appendix

A- 3 -

A-5. Prevalence of DEC pathogroups in water sources

Water samples Pond Water Tape Water Irrigation Water Sewage Water

Total

E. coli Number (n) 14 8 19 31 72

Percentage (%) 19.5 11.1 26.4 43 100

E. coli O157:H7 Number (n) 2 0 3 5 10

Percentage (%) 20 0 30 50 100

Total

Number (n) 16 8 22 36 82

Percentage (%) 19.5 9.8 26.8 43.9 100

A-6. Prevalence of ETEC, EPEC and EHEC in water sources.

E. coli pathotypes Pond Water Sewage Water Running Water Total

ETEC

elt Number (n) 6 10 7 23

Percentage (%) 26.1 43.5 30.4 100

est Number (n) 2 7 4 13

Percentage (%) 15.4 53.8 30.8 100

EPEC

tEPEC Number (n) 3 6 4 13

Percentage (%) 23 46.2 30.8 100

aEPEC Number (n) 1 3 1 5

Percentage (%) 20 60 20 100

STEC stx1/stx2 Number (n) 2 3 5 10

Percentage (%) 20 30 50 100

Appendix

A- 4 -

A-7. Prevalence of diarrheagenic E. coli pathotypes in vegetable sources

Vegetable samples Salad Cucumber Lettuce Spinach Total

E. coli Number (n) 27 23 20 14 84

Percentage (%) 32.1 27.4 23.8 16.7 100

E. coli O157:H7 Number (n) 4 7 3 4 18

Percentage (%) 22.2 38.9 16.7 22.2 100

Total

Number (n) 31 30 23 18 102

Percentage (%) 30.4 29.5 22.5 17.6 100

A-8. Prevalence of ETEC, EPEC and EHEC in vegetable sources

E. coli pathotypes Salad Cucumber Lettuce Spinach Total

ETEC elt/est Number (n) 5 4 2 6 17

Percentage (%) 29.4 23.5 11.8 35.3 100

EPEC bfpA Number (n) 3 1 5 3 12

Percentage (%) 25 8.3 41.7 25 100

STEC stx1/stx2

Number (n) 0 2 1 1 4

Percentage (%) 0 50 25 25 100

Total Number (n) 8 7 8 10 33

Percentage (%) 24.2 21.3 24.2 30.3 100

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