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Molecular Characterization and Antibiotic Susceptibility of Diarrheagenic
Escherichia coli from Children
Tasahel Hamid Al-Dulaimi
* Hawraa Wahab Aziz
* Ali Hussein Al-Marzoqi
*
Saad Abd Al-Aziz
**
Soad Abd Almahdi Mohsin
*
*College of Science for Women, Babylon University
E-mail: ali_almarzoqi@yahoo.co.uk
**College of Medicine, Dhiqar University
Received 21 January 2015 Accepted 11 March 2015
Abstract
In Thi Qar there are relatively few studies have been done to reveal and classify diarrheagenic Escherichia coli
(DEC) strains among children with diarrhea. This study focused genetically at investigating DEC among hospitalized
children under 2 years in Thi Qar city.
A total of 200 children (Below 12 years) with diarrhea and 75 without diarrhea were their stools investigated using
culture on MacConkey and EMB agar, and E. coli isolates were examined for detection of diarrheagenic E. coli types,
antimicrobial susceptibility pattern. Virulence genes detected using PCR. A multiplex PCR system method was used to
detect a species specific gene for E.coli and ten different virulence genes for detection of five pathogroups of DEC
namely enteroaggregative- (EAEC), enteropathogenic- (EPEC), enterotoxigenic- (ETEC), enteroinvasive- (EIEC) and
enterohemorghagic- Escherichia coli (EHEC).
The study has indicated that diarrheagenic E. coli isolates were found mostly in stools of children with diarrhea. DEC
diarrhoeagenic E. coli was significantly detected among diarrheic children (44.8%) compared with control children
(16.4%). Of the DEC pathotypes examined, EAEC was found in (64.73%), ETEC in (19.5%), EPEC in (10.5%) and
EHEC in (5.27%) of diarrheic. Virulence-gene factors in DEC isolated from children with diarrhea and from controls.
The distribution of virulence gene was; pCVD432 93.6%, 87.3%; eaeA 64.2%, 8.5%; bfpA 59.1%, 2.05%; stx1 22.8%,
0.8%; stx2 18.6%, 0%; estA1 heat-stable (ST) 8.14%, 1.3%; estA2-4 heat-stable (ST) 14.6%, 0.2%; eltB heat-labile
(LT) 43.1%, 0.4% in DEC and control children respectively. This study revealed high incidence of diarrheagenic E.
coli isolates and high prevalence of antimicrobial resistance among normal intestinal E. coli with typical EAEC and
typical EPEC predominating. The use of primers for both variants of ST useful to detect the sensitivity for detection of
ETEC strains of hospitalized children in Thi Qar.
Key words: Diarrheagenic, , Escherichia coli, Virulence genes, Phylogenetic grouping, Antibiotic
susceptibility, Thi Qar, DEC, EPEC, EHEC, ETEC, EAEC.
التوصيف الجزيئي والحساسية تجاه المضادات لبكتريا القوقون الاشريكية المسببة للإسھال في الأطفال
الخلاصة
هناك العديد من الد ارسات ذات العلاقه اجريت في ذي قار انجزت حول كشف التصنيف لسلالات بكتريا الإشريكية القولونية Escherichia coli
المت ارفقه مع حالات الاسهال (Diarrheagenic Escherichia coli DEC) في الاطفال المصابين بالاسهال. ركزت هذه الد ارسه على التحري عن
حالات DEC جينيا في الاطفال بعمر دون السنتين لل ارقدين في المستشفيات.
من بين 200 طفل مصابين بحالات الاسهال و 75 طفل سليم (مجموعة سيطره) اخذت عينات الخروج وزرعت على اوساط الماكونكي و الايوسين حيث
تم عزل وتشخيص E. coli كما تمت د ارسة الحساسيه للمضادات الحيويه كما ان جينات الض اروه تم تشخيصها بواسطه تفاعل البلمره المتسلسل PCR
Medical Journal of Babylon-Vol. 12- No. 2 -2015 2015 -الثاني العدد-عشر الثاني المجلد -الطبية بابل مجلة
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وذلك باستخدام بواديء متخصصه لتعيين كل جين وحسب وزنه بلنسبه الى حالات DEC حيث تم تشخيص ,(enteroaggregative (EAEC
.enteropathogenic (EPEC), enterotoxigenic (ETEC), enteroinvasive (EIEC) enterohemorghagic (EHEC)
تم تشخيص حالات DEC في هذه الد ارسه والتي تم عزلها من خروج الاطفال ال ارقدين في المستشفيات حيث كانت النسب 44.8% مقارنه مع حالات
EAEC (64.73%), ETEC (19.5%), EPEC :كالاتي كانت Escherichia coli القولونية الإشريكية مجاميع نسب ان كما ،%16.4 السيطره
pCVD432 93.6%, 87.3%; :كالاتي السيطره وجموعة DEC حالات من لكل اروه الض جينات نسب كانت .(10.5%) and EHEC (5.27%)
eaeA 64.2%, 8.5%; bfpA 59.1%, 2.05%; stx1 22.8%, 0.8%; stx2 18.6%, 0%; estA1 heat-stable (ST) 8.14%, 1.3%;
.التوالي على estA2-4 heat-stable (ST) 14.6%, 0.2%; eltB heat-labile (LT) 43.1%, 0.4%
كشفت هذه الد ارسة عن مدى تاثير عالي من حالات DEC حيث كانت نسب بكتريا E. coli عاليه بمختلف مجاميعها حيث كانت هناك نسبه عاليه من
حالات المقاومه للمضاد الحيوي وبالاخص حالات كل من EAEC و EPEC حيث كانت هي السائده . كان لاستخدام نوعين من البواديء لتشخيص
جين ST مفيدا لغرض تحديد حساسية تشخيص الجين المذكور في حالات الاسهال المرتبطة بـ DEC.
Diarrheagenic, antimicrobial-resistance genes, Escherichia coli, Virulence genes, :الرئيسية الكلمات
Phylogenetic grouping, Antibiotic susceptibility, Thi Qar, DEC, EPEC, EHEC, ETEC, EAEC
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Introduction
iarrhea is one of the major causes of
serious issues among children in the
developing world [1]. In period from
50
th
to 70
th
of last century it was estimated
that more than 4 million children died
annually from diarrhea in developing world
[2,3].
The major etiology of diarrhea was viruses,
bacteria, and parasites. Most common
bacterial causes diarrhea was diarrheagenic
Escherichia coli (DEC) is the most important
etiologic agent of childhood diarrhea and
represents a major public health problem in
developing countries [4]. Identification of
DEC strains requires that these organisms be
differentiated from non-pathogenic members
that constitute normal intestinal flora. DEC
strains can be divided into six main
categories on the basis of distinct molecular,
clinical and pathological features:
enteroaggregative E. coli (EAEC),
enterohemorrhagic (Shiga-toxin producing E.
coli (EHEC/STEC), enteroinvasive E. coli
(EIEC), enteropathogenic E. coli (EPEC),
enterotoxigenic E. coli (ETEC) and diffusely
adherent E.coli (DAEC) [5].
Escherichia coli are the most important
etiological agent of childhood diarrhea and
represent a major public health problem in
developing countries [6].
Accurate detection and identification of
diarrheagenic E.coli can’t be done only by
culture, biochemical and serotyping tests,
since they are indistinguishable from the nonpathogenic
E. coli commonly found in human
feces. Therefore, only DNA based method
such as polymerase chain reaction (PCR)
assay can be used for rapid and reliable
diagnosis, and which has a high sensitivity
and specificity for their detection [7, 8]. The
epidemiological significance of different
DEC pathotypes in childhood diarrhea varies
geographically. Many studies have examined
the clinical relevance of ETEC- associated
diarrhea in certain area [9, 10].
Diarrheagenic Escherichia coli (DEC) are
commonly used as marker of fecal
contamination of waterways in most areas,
which was naturally habitat in the intestinal
tracts of warm-blooded animals [11,34], and
is released into the environment through
deposition of fecal material. In a typical
mixed watershed, host sources of E. coli
may be from humans, farm animals, wildlife,
and pets, among others. These hosts are
generally described as primary habitats, and
until recently E. coli was believed to survive
poorly in the environment, and not to grow in
secondary habitats such as surface water,
sediment, and soil [12,13]. However, it has
been shown that E. coli can survive in the
secondary environments for long periods of
D
Medical Journal of Babylon-Vol. 12- No. 2 -2015 2015 -الثاني العدد-عشر الثاني المجلد -الطبية بابل مجلة
543
time and grow in water, sediment, and soil
even in temperate environments [14, 15].
As we mention previously DEC cannot be
diagnosed adequately by routine method like
culture and biochemical, identification of
these strains is difficult. In contrast,
polymerase chain reaction technique (PCR)
used to detect many numerous genes that
responsible of virulence behavior of DEC
[16,17,18]. Multiplex PCR assays designed
to detect EPEC, ETEC, EAEC, EHEC and
EIEC virulence genes.
The objectives of our study aimed to reveal
the hospitalized children incidence with DEC
diarrhoeagenic E. coli, in addition to
investigate the microbial sensitivity profiles
of E. coli isolates. Phylogenetic isolates of
the DEC was specify of virulence encoding
genes in DEC diarrhoeagenic E. coli isolates
were determined by PCR.
Materials and Methods
Clinical Specimens
During the period from October 2013 and
October 2014, 275 (200 as patients and 75 as
control) stool samples from children were
investigated in Thi Qar to determine the
prevalence of diarrheagenic Escherichia.
Cases and controls were selected from
patients attending the outpatient clinic of
three different hospital