A study of the prevalence of diarrhoeagenic Escherichia
coli in children from Gwagwalada, Federal
Capital Territory, Nigeria
Introduction: Diarrhoeagenic Escherichia
coli (DEC) are major causes of diarrhoea in Nigeria. This study was
conducted to determine the prevalence of diarrhoea caused by DEC within the
Federal Capital Territory, Abuja, Nigeria.
Methods: A total of 730 rectal swabs obtained from
201 children with diarrhoea and 529 healthy controls aged 0-24 months were cultured
for the isolation of Escherichia coli. All E. coli isolates were
investigated by PCR to determine their pathotype.
Results: A total of 61 DEC strains were recovered at
a rate of 18.4% and 2.6% from children with diarrhoea and healthy controls
respectively. The DEC strains recovered were Enteroaggregative
Escherichia coli (34.4%), Shiga-toxin producing Escherichia coli
(31.1%), Enterotoxigenic Escherichia coli
(18.0%), typical enteropathogenic Escherichia coli
(15.0%) and Enteroinvasive Escherichia coli
(1.6%). Shiga-toxin producing Escherichia coli and Enteroinvasive
Escherichia coli were recovered only from children suffering from
diarrhoea and the overall prevalence of DEC strains was significantly higher
among the children with diarrhoea (P < 0.0001). The number of DEC strains
obtained during the dry season was significantly higher (P = 0.012) than the
number obtained in the rainy season.
Conclusion: Diarrhoea caused by E. coli in the
Nigerian children studied is associated with several diarrhoeagenic
pathotypes and a significant proportion of the
healthy children were found to harbour EAEC and ETEC strains. These
asymptomatic carriers may be regarded as potential transmitters of infection to
vulnerable children in the study area.
Diarrhoeal diseases are a
global public health problem causing considerable morbidity and mortality among
infants and children especially in the developing countries [1-2].
Diarrhoea is responsible for annual global deaths of about 2.6 million people
mostly among African children under the age of 5 years 
and the significant reduction in child mortality observed in recent years has
been attributed to the practice of exclusive breast feeding as well as recourse
to oral rehydration therapy in the treatment of diarrhoea .
Infections due to faecal-water contamination are
caused by a host of enteric infectious agents including bacteria, viruses,
fungi and parasites. Among the bacteria, at least six pathogenic of diarrhoeagenic Escherichia coli (DEC strains) are
frequently detected worldwide. These pathotypes have
been described based on the genes mediating the virulence factors associated
with the diarrhoeal disease caused by them: Enteropathogenic
E. coli (EPEC), Enterotoxigenic E. coli
(ETEC), Shiga toxin-producing E. coli (STEC) or Enterohaemorrhagic
E. coli (EHEC), Enteroaggregative E. coli
(EAEC), Enteroinvasive E. coli (EIEC) and diffusely
adherent E. coli (DAEC) [5-6].
The identification of DEC pathotypes
is through the use of molecular methods for the detection of the genes
responsible for mediating the substances responsible for pathogenicity.
This is essential for identification and classification of DEC and is based on
the presence of different chromosomal and/or plasmid-encoded virulence genes
that are absent in commensal E. coli.
Moreover, the prevalence and other epidemiological features of these pathogens
as causative agents of diarrhoea vary from one region to the other, and even
between and within countries in the same geographical area [7-8].
In Nigeria for example, only very few studies have investigated the
microbiology of childhood diarrhoea and these have all been carried out in the
South. This is a report of such a study carried out in the North-Central part
of the country.
This study therefore investigated the prevalence
of diarrhoeagenic Escherichia coli isolated
from children in Gwagwalada, Federal Capital
Territory, Nigeria as a means of determining the distribution of DEC associated
diarrhoea in the North-Central part of Nigeria.
A total of 730 children aged 0-24 months who
reported to the paediatric unit of the University of Abuja Teaching Hospital, Gwagwalada with complaints of diarrhoea (with or without
fever or other accompanying symptoms) and had not taken any antimicrobial agent
in the preceding week were recruited into this study over a period of one year
(1st April, 2008 to 31st March, 2009). The children in the same age range who
reported for the immunization programme at the Township Clinic in the same town
were regarded as apparently healthy children and used as controls in this
study. Gwagwalada is a fast growing satellite town in
the Federal Capital Territory, Abuja in the North Central Nigeria and the
teaching hospital is a tertiary care hospital with a 500-bed capacity that
offers a full range of services to people living mostly in the Middle Belt area
of the country. The study was approved by the ethical committees of the
participating institutions while the parents or guardians of the children gave
informed consent and filled a questionnaire to provide demographic data and the
breast-feeding pattern for each child.
Faecal specimens were obtained as rectal swabs
from each of the children in the study by inserting sterile cotton wool
applicators to the rectums of the children. The faecal material thus obtained
was transported to the laboratory in sterilized Cary Blair transport medium for
the culture and isolation of E. coli. Rectal swabs were used in this
study because they are more easily obtained from all the volunteers than the
stool samples which are considered the gold standard for the identification of
gastrointestinal (GI) tract colonization with bacteria. Moreover, Lautenbach et al.  have shown that
rectal swabs are able to allow the cultivation of 90 - 100% faecal contaminants
when compared to the faecal samples in the isolation of enteropathogens.
Isolation and storage of E. coli
Each rectal swab from the subjects was
inoculated onto the surface of MacConkey and Eosin Methylene Blue (EMB) agars (Oxoid,
UK) within 24 hours of collection and streaked for isolated colonies. The
organisms that produced characteristic discrete colonies after 24 hours of
incubation at 37°C were streaked onto fresh sterilized Nutrient agar (Oxoid, UK) and identified by conventional biochemical IMViC reactions (Indole, Methyl
red, Voges Proskauer,
Citrate, Urease tests) . The
isolates that were positive to indole and methyl red
tests but negative to voges proskauer,
citrate and urease tests were identified as E.
coli. All E. coli colonies from a single rectal culture with
identical colony morphology, and biochemical properties were assumed to be
identical while four to five E. coli isolates with different colony morphology
that were positive to the conventional biochemical tests arising from a single
rectal swab from each of the children were maintained in the laboratory in cryovials (Nalgene, USA) by
cryopreservation at -70°C and also stored in nutrient agar slants at 4°C in a
refrigerator for the investigation of the genes encoding pathogenicity
at molecular level.
Screening for Diarrhoeagenic
E. coli genes
DNA from each confirmed E. coli isolate was
extracted from the organism after a 24 hour incubation period on nutrient agar
plates by suspending three colonies in 50 µl of deionized
water and boiling for 10 minutes. This was cooled on ice for at least 2
minutes, followed by centrifugation at 3 RAM (3,000 per g for 5 minutes) using
BIO-RAD Microcentrifuge (Model 16K) (BIO-RAD
Laboratories, USA) to pelletise the cell debris. Exactly
2 µl of each test isolate’s supernatant was used as the DNA template for
Polymerase Chain Reaction (PCR) analysis. Two (2) µl of lysate
from the reference strains EPEC E2348/69, EAEC O42, ETEC H10407, EIEC EDL1284,
and STEC EDL931 which served as positive controls and E. coli K-12
DH5α which was the negative control and from all the E. coli-positive
isolates were subjected to multiplex PCR with specific
primers. This was with a view to detecting the following diarrhoeagenic
virulence markers: eaeA (structural gene for intimin of EHEC and EPEC), bfpA
(structural gene for the bundle-forming pilus of
EPEC), stx1 and/or st×2 (verocytotoxin 1 and 2 of
EHEC and STEC), eltB and/or estA (enterotoxins LT and
ST of ETEC), ipaH (invasion-associated locus
of the invasion plasmid found in EIEC and Shigella)
and pCVD (the nucleotide sequence of the EcoRI-PstI DNA fragment of pCVD432 of EAEC) as described by
Nguyen et al. and Aranda et al. [7,11].
PCRs were performed using an optimized protocol
which was carried out on ice with a 100 µl reaction mixture containing 10 mM Tris-HCl (Trisaminomethane
hydrochloride) (10× Standard Reaction Buffer (pH 8.3) (New England Biolabs Inc. UK), 50 mM MgCl2
(New England Biolabs Inc. UK), 10 mM
concentration of dNTPs (Madison, USA), 50 nmol each of PCR primers (Eurofins,
USA), a 5 units per µl pure Taq DNA polymerase
(Standard Taq Mg-free Buffer) (New England Biolabs Inc. UK) and 2 µl of the DNA template. The optimal concentration
of each primer pair in the reaction mixture was determined empirically. The PCR
was carried out in a GeneAmp PCR system 9700 (Applied
Biosystems, Singapore) with the following thermocycling conditions: For PCR assay designated as
"1"_ 50°C (2 min, 1 cycle); 95°C (5 min, 1 cycle); 40 cycles of 95°C
(40 s), 58°C (1 min), and 72°C (2 min); and a final extension step at 72°C (7
min, 1 cycle); and for PCR assay designated as "2" (which was divided
into PCR 3 and PCR 4) _ 50°C (2 min, 1 cycle); 95°C (5 min, 1 cycle); 40 cycles
of 95°C (45 s), 50°C (1 min), and 72°C (1 min); and 72°C (7 min, 1 cycle) in a
thermal cycler (GeneAmp PCR system 9700, Applied Biosystems, Singapore). The PCR products (10 µl) were
evaluated on a 1.5% (w/v) agarose gel (UltraPure Agarose; Invitrogen Life Technologies) at 100 mV for 60 minutes
using BIO-RAD Power Pac 3000 (BIO-RAD Laboratories, USA) and a molecular weight
marker (100 bp DNA Ladder; New England Biolabs. UK) was run concurrently. The DNA bands were then
visualized and photographed under UV light (using UVitec,
Cambridge UK) and Video copy Processor; Mitsubishi Electro, Malaysia) after
staining the gel with ethidium bromide.
Frequencies and percentages were calculated for
the study variables and the data obtained were compared with the use of a
two-tailed Chi square and Fisher's exact tests. A P-value of less than or equal
to 0.05 (P ≤ 0.05) was considered to be statistically significant.
A total of 730 rectal
swabs from 201 children with diarrhoea and 529 apparently healthy of ages 0-24
months that were made up of 337 females and 393 males were examined for the
presence of E. coli strains. Only 277 and 388 E. coli isolates
recovered from the screened specimens from diarrhoea and control groups
respectively were screened for possible virulent diarrhoeagenic
genes. The analysis of the questionnaire on feeding pattern of all the children
revealed that 76% of the children in age group 0-3 months were exclusively
breast fed (Table 1).
The diarrhoeagenic E.
coli strains were detected at an overall rate of 18.4% (n=51) and 2.6%
(n=10) from the isolates recovered from diarrhoea and control groups
respectively. They comprised of Enterotoxigenic Escherichia
coli (n =11), Typical enteropathogenic
Escherichia coli (n =9), Enteroaggregative Escherichia
coli (n =21), Shiga-toxin producing Escherichia coli (n =19) and Enteroinvasive Escherichia coli (n =1) (Table 2). The overall prevalence of DEC
strains was significantly higher among the isolates from the children with
diarrhoea (P < 0.0001).
The highest proportion of DEC (15.4%) was
detected among the children in age group 4-6 months whilst those in age groups
0-3 months and 22-24 months provided the lowest number of DEC isolates. The
EPEC strains were found only among the children in age group 0-9 months whilst
ETEC strains were most frequently encountered among the children aged 16-18
months. A total of 87.5% of EAEC and 78.9% of STEC strains were significantly
found among children older than 3 months (P < 0.00001) in the children with
The detection of DEC in the study year was found
to be highest in March (27.1%), moderate in May (14.5%) and December (15.0%)
but lowest in June with no DEC strain recovered. The observed difference in the
monthly prevalence of the DEC was highly significant in the month of March (P =
0.00002) (Table 3). The ETEC and EPEC
strains were significantly detected in the dry season (P = 0.003 and 0.015,
respectively) and the overall prevalence of diarrhoeagenic
E. coli was significantly higher during the dry season (November-March)
than the rainy season (April-October) (P = 0.012) (Table
coli strains are pathogens of public health importance affecting both
adults and children worldwide. Most diarrhoeal cases in children under 5 years
of age have been shown to be due to diarrhoeagenic E.
coli in which ETEC, EAEC and EPEC strains are the most prevalent in the
developing countries [8, 12].
In this study, the frequency of recovery of DEC
strains was significantly higher among the children with diarrhoea (18.4%) than
those without diarrhoea (2.6%). This significant association of DEC with
diarrhoea in the study environment is as it has been reported by other previous
studies carried out in other locations such as Ghana 
and Brazil . Similar results have also been reported
from the south eastern part of Nigeria . This study
however recorded a lower rate of recovery of DEC from the diarrhoeal children
than these other studies where the rate of recovery ranged between 21.4% in
India  and 36.8% in Brazil .
The low prevalence of DEC among the diarrhoeal cases also suggests that other
causative agents (such as rotavirus, protozoan Giardia,
Salmonella and Shigella species)
that were not investigated in this study might be other causes of the diarrhoea
[17,18]. The recovery of DEC from
only 2.6% of the apparently healthy subjects in this study environment suggests
that these pathogenic organisms are rarely encountered in healthy children and
the few from which the diarrhoeagenic E. coli
were isolated might be recovering from diarrhoea or were in the pre-symptomatic
stage of the infection.
The distribution of DEC among the various
children age groups in this study showed that children in age groups 0-3 and
22-24 months had the lowest prevalence of DEC infections. This observation
suggests that the children were protected in the first three months of their
life, when they were mostly being breast fed, by the antibodies in breast milk
and later on by their own acquired immunity [19, 20]. The prevalence of DEC was therefore found to be highest
among the children in age groups 4-9 and 16-21 months age groups in which
immunity conferred by breast milk had been waned before their capacity to mount
an effective immunological response is developed [21-22].
In addition, children within the age 16 to 21 months were found to have been
weaned and their exposure to adult foods constitutes a risk factor since such
foods are more likely to be contaminated by microorganisms than breast milk.
The results of this study showed a seasonal
variation in the prevalence of DEC infection in the environment. It was
observed that the overall prevalence of infection was significantly higher
during the dry season than the rainy season (P = 0.012). The peak prevalence
was observed in March, a period considered as one of the driest and hottest
months in the study area, when there is a shortage of potable water in area in
which municipally treated water is not available. In addition, the high
temperatures which are characteristic of this period are favourable to the
proliferation of infectious agents in the tropics .
This observation is similar to previous studies on seasonal variation of DEC
infection reported by El Metwally et al. in Egypt  and Albert et al. in Kuwait . The
four common pathotypes of E. coli which include ETEC,
EPEC, STEC and EAEC were detected in this study. Their frequencies of recovery
were significantly higher in the diarrhoea group than in the non-diarrhoeal
category. This supports the findings of other investigators in some other
developing countries [7,13,16,24].
Enteroaggregative E. coli (EAEC) strains were the most
frequently recovered DEC pathotypes in this study.
Their prevalence was found to be significantly higher among the children with
diarrhoea than those without diarrhoea (P = 0.0014) and the children in the 0-3
month age group were the least affected (12.5%). EAEC was the pathotype with the highest prevalence (50.0%) among the
children without diarrhoea and 80% of the children in these categories were
from the 0-6 month age group. This observation shows EAEC as an increasingly
recognized cause of diarrhoea illness among children in developing countries as
reported by other workers [14,16,26]. The high rate of the isolation
of EAEC in this group of subjects and the significantly higher prevalence
(80.0%) among the 0-6 month age group without diarrhoea suggests that immunity
to this pathotype is widely developed at an early age
and supports the postulation of Okeke  that infection with EAEC before weaning was unlikely to
lead to diarrhoea.
Shiga toxin-producing Escherichia coli
(STEC) infection causes acute and bloody diarrhoea. The STEC strains recovered
in this study were made up of 8 strains with St×1, 3 with St×2 and 8 with both
St×1 and St×2 genes. They were only detected from the isolates from children
with diarrhoea at a prevalence rate of 6.9% and were frequently found in
children within the 6-24 month age group. This observed prevalence of STEC
strains disagrees with the findings of previous workers who reported very low
or no isolation in children with diarrhoea [15,16,24] while it is similar to reports of
Alikhani et al.  in Iran and
Garcia et al.  in Brazil who recovered STEC of 8.7%
and 7.4% respectively from children with diarrhoea. The reason for the
isolation of this pathotype within the study
environment is not known and requires further investigation.
The ETEC strains in this study were made up of 7
strains bearing genes encoding LT, 3 strains with ST and 1 strain with both LT
and ST (two enterotoxins). The ST strains were only
found in the diarrhoea group while the only isolate that produced both LT and
ST was from an apparently healthy child. This higher prevalence of LT-ETEC over
ST-ETEC observed in the children of ages 0-24 months has been previously
reported by Valentiner-Branth et al.  in Guinea and El Metwally et
al.  in Egypt while the detection of ETEC strains
producing the two enterotoxins (LT and ST) have been
reported by Okeke  in
Ile-Ife, Nigeria and El Metwally et al.  in Egypt. ETEC diarrhoea in this study was encountered
mainly in children in the 4-18 month age group, whilst none of the children in
the 0-3 month age group had ETEC diarrhoea but harboured the LT and ST strains.
The high prevalence of ETEC diarrhoea among the older children may be due to
the fact that children in these groups lose the immunity conferred on them
through the antibodies passed to them from the breast milk of their mothers.
Enteropathogenic E. coli are currently classified into
two sub-categories, that is, typical and atypical EPEC. While typical EPEC are
established pathogens, the pathogenicity of atypical
EPEC is still a subject of debate . The EPEC strains
detected in this study were all typical EPEC and they were found to be
significantly higher among children with diarrhoea than those without
diarrhoea. Of the 9 isolates of EPEC recovered, only one of them was found in a
child without diarrhoea confirming the association between typical EPEC and
diarrhoeal disease. The only EPEC strain in the non-diarrhoeal group was found
in a 6 month old child whilst one of the strains in the diarrhoea group came
from a 3 month old child. This shows the protective power of mother's partial
immunity and exclusive breastfeeding on the children against EPEC diarrhoea . All the EPEC strains detected in this study were from
the children in the 0-9 month age group thus suggesting that EPEC is one of the
main causes of infantile diarrhoea .
The isolation of 1(0.6%) EIEC isolate from
diarrhoea group in this study agrees with previous studies in the detection of
very low rate of this pathotype that is known to cause
diarrhoea symptoms similar to shigellosis in adults and children [14,24]. The
lack of epidemiological attention to EIEC is related to the low incidence of
this pathogen as a cause of diarrhoea when compared to other pathotypes of diarrhoeagenic E.
coli. As pointed out by Vierra et al. , researchers have not reported the isolation of these
organisms from patients with diarrhoea. One of the reasons why the isolation
rate of this type of DEC is low is related to the fact that they are missed
when only lactose fermentation is used as a preliminary screening tool for diarrhoeagenic E. coli since over 70% E. coli
in this group do not ferment lactose .
The findings from this study corroborate the
reports in literature on the protection conferred on infants by breast-milk
against infectious diseases. Breast-milk is considered to be the best source of
nutrients and immunological factors needed for infants to grow and resist
infections in the early stages of life . The practice
of exclusive breastfeeding (EBF) in the first 6 months of life and continued
breastfeeding up to the 11th month of birth has been identified as the single
most effective preventive intervention in reducing child mortality due to
diarrhoea and respiratory tract infections . Hence the
recommendation that EBF should be practiced by nursing mothers globally. The
results of this study however showed that in the Federal Capital Territory of
Nigeria, only 62.5% of infants in the age bracket 0-6 months were exclusively
fed on breast-milk and only 17.4% of children above the age of 6 months were
still being breast fed. The figure of 62.5% for exclusive breast feeding, in
this study, whilst lower than the desired figured is however well in excess of
16.4% and 32.2% which have been reported from other parts of northern Nigeria [36,37]. Other studies in Nigeria also
show that compliance with exclusive breast feeding is significantly lower than
Given the effectiveness of exclusive breast feeding to the reduction of infant
morbidity and mortality due to diarrhoea; it is important that efforts to
impress desirability of breast feeding on mothers be intensified and compliance
monitored throughout Nigeria and indeed other developing countries.
The results of this study
suggest that diarrhoeagenic E. coli,
especially STEC, EAEC and EPEC are strongly associated with childhood diarrhoea
within the study environment whilst both EAEC and ETEC strains were recovered
from apparently healthy children who could then be sources of the transmission
of these pathogens to other children. Such transmission could however be
prevented through the widespread application of hygienic practices, the
training for which should be carried out by appropriately trained public health
The authors declare no
conflicts of interest.
Awolowo University) conceptualized; designed and
coordinated the research. AO (Niger Delta University) acquired the data,
performed the microbiological testing and molecular experiments and drafted the
manuscript. OI and AL (Obafemi Awolowo
University) critically revised the paper for important intellectual content and
all authors approved the final version.
We thank the management
and staff of the institutions where the samples were collected for their
support and technical assistance, Drs OA Aboderin and
WB Odetoyin of the department of Medical Microbiology
and Parasitiology, Obafemi Awolowo University, Ile-Ife, for the helpful discussions
and Prof. IN Okeke of Haverford College, USA, for the
provision of materials used for molecular studies.
Table 1: Analysis of the feeding pattern
of children in the breast feeding age groups among the subjects in the study
Table 2: Frequency of Diarrhoeagenic E.
coli among the diarrhoea group and other childrens
categories in the study centre
Table 3: The monthly trend of diarrhoeagenic E.
Table 4: Seasonal variation of the diarrhoeagenic E.
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