prevalence and molecular characterization of...
TRANSCRIPT
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
S. No Title Page. No
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
S. No Title Page. No
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
19 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.
83
20 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.
83
21 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
84
22 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
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.
This catastrophe was the biggest after 2005 earthquake, which left people of affected
areas in a horrible situation. According to World Health Organization (WHO) 2010
report, flooding has affected 21 million people. Flooding cause 5.3 million medical
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
Chapter 2 Review of Literature
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).
Chapter 2 Review of Literature
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
Chapter 2 Review of Literature
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.
Chapter 2 Review of Literature
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).
Chapter 2 Review of Literature
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
Chapter 2 Review of Literature
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
Chapter 2 Review of Literature
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
Chapter 2 Review of Literature
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).
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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
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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).
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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
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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).
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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
Chapter 2 Review of Literature
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
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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,
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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
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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
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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
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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
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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
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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).
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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
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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
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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
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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.
This catastrophe was the biggest after 2005 earthquake, which left people of affected
areas in a horrible situation. According to World Health Organization (WHO) 2010
report, flooding has affected 21 million people. Flooding cause 5.3 million medical
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).
Chapter 2 Review of Literature
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
Chapter 2 Review of Literature
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).
Chapter 2 Review of Literature
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,
Chapter 2 Review of Literature
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).
Chapter 2 Review of Literature
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
Chapter 2 Review of Literature
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).
Chapter 2 Review of Literature
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.
Chapter 3 Materials and Methods
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
Chapter 3 Materials and Methods
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
Chapter 3 Materials and Methods
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
Chapter 3 Materials and Methods
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.
Chapter 3 Materials and Methods
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
Chapter 3 Materials and Methods
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).
Chapter 3 Materials and Methods
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),
Chapter 3 Materials and Methods
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).
Chapter 3 Materials and Methods
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.
Chapter 3 Materials and Methods
62
Table 3. Oligonucleotide sequence of primers used for lineage specific polymorphism
Assay
PRIMER
(LSPA 6)
5' --> 3' primer
Forward
5' --> 3' primer
Reverse
Source Target/comment
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
Source Target/comment
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
Chapter 3 Materials and Methods
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
stx1 prophage detection 702
wrbA
insertion site
region
AGGAAGGTA
CGCATTTGAC
C (primer C)
CGAATCGCTA
CGGAATAGAG
A (D)
Besser et al.,
2007 and
Shaikh et al
2003
presence of stx2 prophage 314
wrbA right
junction
AGGAAGGTA
CGCATTTGAC
C (primer C)
ATCGTTCGCA
AGAATCACAA
(primer G)
Besser et al.,
2007 and
Shaikh et al
2003
stx2 prophage detection 506
wrbA left
junction
CCGACCTTTG
TACGGATGTA
A (H)
CGAATCGCTA
CGGAATAGAG
A (D)
Besser et al.,
2007 and
Shaikh et al
2003
stx2 prophage detection 592
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
Chapter 3 Materials and Methods
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
presence of stx2 prophage 208
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
Chapter 5 Discussion
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
Chapter 5 Discussion
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
Chapter 5 Discussion
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
Chapter 5 Discussion
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).
Chapter 5 Discussion
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%
Chapter 5 Discussion
92
(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).
Chapter 5 Discussion
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
Chapter 5 Discussion
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
Chapter 5 Discussion
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).
Chapter 5 Discussion
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).
Chapter 5 Discussion
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
Chapter 5 Discussion
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
Chapter 5 Discussion
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
Chapter 5 Discussion
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
Chapter 5 Discussion
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
Chapter 5 Discussion
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
Chapter 5 Discussion
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
Chapter 5 Discussion
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
Chapter 5 Discussion
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.
Chapter 5 Discussion
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