Some clinical isolates of enteropathogenic Escherichia
coli (EPEC) lack bundle-forming pili and are termed atypical
EPEC. The aim of this study was to determine if atypical
EPEC are pathogens by comparing the clinical features of
patients infected with atypical EPEC with those of children
infected with other causative agents of diarrhea. Fecal sam-
ples obtained from children attending the Royal Children’s
Hospital in Melbourne for investigation of diarrhea were
examined for adenovirus, rotavirus, Campylobacter spp.,
Salmonella spp., protozoa, and pathogenic E. coli. Clinical
data were obtained by using a standardized pro forma and
analyzed separately. Patients infected with atypical EPEC
experienced mild, nondehydrating, and noninflammatory
diarrhea that was not particularly associated with fever,
vomiting, or abdominal pain. However, the duration of diar-
rhea in patients infected with atypical EPEC was significant-
ly longer than that caused by the other species or where no
pathogens were identified. Infection with atypical EPEC is
associated with prolonged diarrhea.
ing to their virulence determinants (1,2). The specific
nature of these virulence determinants imbues each patho-
type with the capacity to cause clinical syndromes with dis-
tinctive epidemiologic and pathologic characteristics (2).
For example, enterotoxigenic E. coli causes watery diarrhea
in children in developing countries and in travelers to those
countries, whereas enterohemorrhagic E. coli (EHEC) may
cause hemorrhagic colitis and the hemolytic uremic syn-
drome because of the production of Shiga toxins.
Enteropathogenic E. coli (EPEC) shares several key viru-
lence determinants with the most common varieties of
EHEC but does not produce Shiga toxins nor cause hemor-
he varieties of Escherichia coli that cause diarrhea are
classified into pathogenic groups (pathotypes) accord-
rhagic colitis or hemolytic uremic syndrome. Instead, it
causes nonspecific gastroenteritis, especially in children in
developing countries (3,4). EPEC also differs from other
pathotypes of E. coli in that it typically carries an EPEC
adherence factor plasmid (pEAF). This plasmid encodes 1)
bundle-forming pili (Bfp), which promote bacterial adher-
ence to epithelial cells and are an essential virulence deter-
minant (5), and 2) a transcriptional activator, Per, that
upregulates genes within a chromosomal pathogenicity
island, termed the locus for enterocyte effacement (6,7).
This pathogenicity island encodes a number of essential
virulence proteins, including the surface protein intimin
(the product of the eae gene), which is required to produce
the attaching-effacing lesions that are a key feature of
EPEC-induced pathology. A subset of EPEC, known as
atypical EPEC, do not carry pEAF and hence do not pro-
duce Bfp or Per (4). Accordingly, their role in disease is
controversial. Recently, we and others investigated the
causes of community-acquired gastroenteritis in Melbourne
(8,9). Among the infectious agents that were sought in these
studies was atypical EPEC, which emerged as the single
most frequent pathogen in the study population (9).
To determine if atypical EPEC are also responsible for
diarrhea in hospitalized children, we undertook a compre-
hensive microbiologic study of patients with diarrhea at
the Royal Children’s Hospital in Melbourne.
Patients and Methods
Patients were children with diarrhea attending the
Royal Children’s Hospital, Melbourne, between March 1
and August 31, 2003. They were considered for inclusion
Atypic al Enteropathogenic
Escherichia c oli Infec tion and
Prolonged Diarrhea in Children
Rang N. Nguyen,*1Louise S. Taylor,*† Marija Tauschek,*† and Roy M. Robins-Browne*†
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 4, April 2006597
*University of Melbourne, Parkville, Australia; and †Murdoch
Children’s Research Institute, Parkville, Victoria, Australia
1Current affiliation: An Giang General Hospital, Long Xuyen City,
An Giang, Vietnam
in the study when an obviously loose stool sample from a
child <14 years of age was received at the Diagnostic
Microbiology Laboratory for investigation. After their
caregivers, attending physicians, and medical records had
been consulted, patients were considered eligible for inclu-
sion in the study if, during the current illness, they had
passed >3 loose stools within a day or had experienced
loose stools plus vomiting, abdominal pain, or rectal bleed-
ing. Patients with chronic gastrointestinal disorders, such
as inflammatory bowel or celiac disease, were excluded, as
were those with cystic fibrosis, leukemia, and other
immunosuppressive disorders. Repeat samples and sam-
ples from children who had received antimicrobial agents
within the preceding week were also excluded.
Clinical data were obtained in accordance with a stan-
dardized pro forma and were analyzed before the results of
the laboratory findings were known. Data collected includ-
ed age; gender; date of onset of illness; symptoms and clin-
ical signs, including characteristics of stools, abdominal
pain, vomiting (number per day and duration), fever,
abdominal tenderness, largest number of bowel move-
ments in a 24-hour period preceding the sample collection,
and extent of dehydration. Duration of diarrhea was esti-
mated from the passage of the first loose stool to the
patient’s last appearance in the ward or 1 day after dis-
charge. Patients with temperatures of >38°C, taken by
tympanic thermometer, were considered febrile. Severity
of illness was estimated by using the 20-point scale devel-
oped by Ruuska and Vesikari (10).
All stool specimens were the first specimen obtained
from a patient on a hospital visit, and specimens were
investigated within 4 hours of collection. Specimens were
examined macroscopically for color and consistency and
by light microscopy for leukocytes, erythrocytes, and par-
asitic forms (amebas, cysts, and ova) by using a saline-
and-iodine wet preparation and a modified Ziehl-Neelsen
stain for oocysts of Cryptosporidium spp. (11). Samples
were tested by enzyme immunoassay for enteric aden-
oviruses and rotaviruses and cultured for E. coli,
Salmonella, Shigella, Yersinia, and Campylobacter spp.
To reduce the cost of the investigation, diarrheogenic
strains of E. coli were sought only during the first 11
weeks of the study, from March 1 to May 15. Bacteria
were isolated from fecal samples by direct plating on
MacConkey agar (Oxoid Ltd., Basingstoke, UK). After
overnight incubation at 37°C, a sterile cotton swab was
used to transfer the entire growth from each plate into
Luria broth containing 30% (vol/vol) glycerol, which was
then frozen at –70°C until required. E. coli pathotypes
were identified by polymerase chain reaction (PCR) and
confirmed by Southern hybridization (9). Briefly, template
DNA for use in PCR was prepared from bacteria isolated
from MacConkey agar plates and grown in 2.5 mL
MacConkey broth with shaking at 37°C overnight.
Bacteria from this culture were washed in phosphate-
buffered saline, resuspended in sterile distilled water, and
heated for 10 min at 100°C. Samples were then placed on
ice for 5 min and recentrifuged for 5 min at 16,000 × g.
Aliquots of the supernatant were pipetted into sterile tubes,
stored at –20°C for <1 week, and then diluted 1 in 10 in
distilled water before being added to the PCR mix. PCR
amplifications were performed in a GeneAmp PCR
System 9700 thermal cycler (Applied Biosystems, Foster
City, CA, USA) with the PCR primers and conditions
described previously (9). Genes identified by these primers
and their association with each pathotype of diarrheogenic
E. coli are listed in Table 1. PCR for the lacZ gene, which
is present in almost all wildtype strains of E. coli, was
included as a control to ensure that negative PCR results
were not due to the absence of viable bacteria in the sam-
ple or the presence of inhibitors in the reaction mixture.
Samples that were PCR negative for lacZ were excluded
from further analysis. At the conclusion of the PCR, 10 µL
of the reaction mixture underwent electrophoresis on 2.5%
96-well format agarose gels (Electro-fast; Abgene, Epsom,
UK). Gels were stained with ethidium bromide, visualized
on a UV transilluminator, and photographed. A portion of
the PCR product was retained for Southern blotting, which
was performed by using capillary transfer of separated
DNA fragments onto positively charged nylon membranes
(Roche Diagnostics Ltd., Lewes, UK). Digoxigenin-
labeled DNAprobes were prepared from the control strains
of diarrheogenic E. coli listed in Table 1 and used as
described (9). PCR- and probe-positive bacteria were
assigned to a pathotype according to the criteria in Table 1.
Equivocal or ambiguous assays were repeated, and if still
unclear, were excluded from further analysis. Atypical
EPEC strains were isolated in pure culture from the origi-
nal sample and then serotyped by using hyperimmune rab-
bit antisera to O-antigens O1 through O181 (18). These
strains were also subjected to PCR to determine the intimin
subtype and to investigate the presence of selected viru-
lence-associated genes by using the PCR primers and con-
ditions described previously (9).
Statistical analysis of quantitative and qualitative data
was performed by using InStat, Version 3.05 (GraphPad
Software Inc., San Diego, CA, USA). A2-tailed p value of
<0.05 indicated statistical significance. For the analysis of
clinical features associated with infection, patients whose
stools yielded >1 pathogen were excluded.
598 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 4, April 2006
Frequency of Viral, Parasitic, and Bacterial Pathogens
After exclusion of repeat samples and samples from
patients >14 years of age or with cystic fibrosis, chronic
inflammatory bowel disease, leukemia, or a history of
recent antimicrobial drug usage, 303 of 972 consecutive
fecal samples remained for analysis. Of these, 134 were
from the first period of the study, March 1–May 15, when
diarrheogenic E. coli were sought together with other
enteropathogens, and 169 were from the period May
16–August 31, when E. coli were not sought.
The frequency of bacterial, viral, and parasitic
pathogens identified during the 2 phases of the study are
shown in Table 2. During the first period, a putative etio-
logic agent was identified in 88 (66%) of 134 children.
Diarrheogenic E. coli were found in 42 (31%) of these
children, followed by enteric adenovirus (10%),
Salmonella sp. (10%), Campylobacter spp. (9%), Giardia
sp. (6%), rotavirus (4%), and Cryptosporidium sp. (2%).
Of the 42 E. coli isolates, 30 (71%) were EPEC; 6 (14%)
were Shiga toxin–producing E. coli (STEC), of which 3
were EHEC; 4 (10%) were enteroaggregative E. coli
(EAEC), 1 (2%) was enterotoxigenic E. coli; and 1 (2%)
was enteroinvasive E. coli. Nine children (7%) were
infected with >1 pathogen, including 2 concurrently infect-
ed with EPEC and adenovirus or EPEC and rotavirus, and
1 each with EPEC and Giardia sp.; STEC and
Campylobacter sp.; STEC and Giardia sp.; EAEC and
Campylobacter sp., and EAEC and rotavirus.
All EPEC isolates were atypical EPEC (i.e., PCR neg-
ative for bfpA). Determination of the O:H serotype and
intimin subtype of 29 of the 30 EPEC strains (1 was not
viable) indicated that they were highly heterogeneous
(Table 3). Although 3 strains (R41, R151, and R446) were
O-nontypable:H34, intimin-α2; and 2 (R89 and R104)
were O153:H7, intimin β, these isolates were neither tem-
porally nor geographically related to each other and
showed some differences in their carriage of accessory vir-
ulence-related factors (data not shown). Two other isolates
(R250 and R436) were O33:H6 but had different intimin
types. Ten isolates were O-serogroups that were classified
as nontypable because they did not react with any of the
available O-typing sera (O1–O181), and 2 isolates could
not be serotyped because they were rough. Only 1 isolate
(R404) belonged to an E. coli serotype, O128:H2, that is
commonly associated with EPEC (4).
During the second period of the study, when E. coli was
not sought, putative pathogens were identified in 99
(58.6%) of 169 children; rotavirus was the most frequent
(33.7%), followed by Campylobacter (11.8%), adenovirus
Cryptosporidium (0.6%) spp. Four patients were infected
with >1 pathogen: 3 concurrently infected with rotavirus
Atypical Enteropathogenic Escherichia coli Infection
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 4, April 2006 599
and adenovirus and 1 with adenovirus and Salmonella sp.
Shigella and Yersinia spp. were not identified during either
period of the study. The frequency of rotavirus infection
during the second phase of the study was significantly
greater than during the first phase (odds ratio [OR] 13.13;
95% confidence interval [CI] 5.08–33.91, p<10–6, 2-tailed
Fisher exact test), confirming the well-known association
of rotavirus with winter diarrhea (12). The frequency of
patients in whom no pathogens were identified was not
significantly different between the 2 study periods (OR
0.74, 95% CI 0.46–1.18, p>0.2, 2-tailed Fisher exact test),
despite the omission of tests for diarrheogenic E. coli dur-
ing the second period. This finding suggests that E. coli did
not account for a large number of cases during the second
period of the study and accords with our previous observa-
tions that diarrhea due to E. coli is relatively less frequent
during winter (9,19).
Comparison of Clinical and Laboratory Findings
The clinical and laboratory features of patients infected
with different pathogens were compared (Table 4). Patients
infected with >1 pathogen were excluded from this analy-
sis, as were those infected with Giardia
Cryptosporidium spp. or diarrheogenic E. coli other than
EPEC because their numbers were too small for the results
to be meaningful. For this analysis, only those patients in
whom no pathogens were identified from the first study
period were considered because of the possibility that
some of those studied during the second period were
infected with diarrheogenic E. coli.
Patients infected with EPEC were of a similar age
(median 16.9 months) to those from whom no pathogens
were isolated (median age 11.6 months). Of the various
groups of patients defined according to the cause of diar-
rhea, only those infected with Campylobacter spp. (medi-
an age 34.2 months) differed significantly in age from
those with EPEC (p = 0.0002, Mann-Whitney U test).
Eighteen (72%) of 25 children monoinfected with EPEC
were boys compared with 20 (44%) of 46 children in
whom no pathogens were identified (OR = 3.34, 95% CI
1.17–9.55, p = 0.03, 2-tailed Fisher exact test), and with 49
(47%) of 104 children enrolled in phase 1 of the study who
were not infected with EPEC (OR = 2.89, 95% CI
1.11–7.5, p = 0.03).
Patients infected with rotavirus or adenovirus were sig-
nificantly more likely to have a history of vomiting than
those with no pathogen identified or those infected with
EPEC, Salmonella, or Campylobacter spp. The frequency
of vomiting in patients infected with EPEC and in those
with no pathogen identified was similar. Abdominal pain
was reported significantly more frequently in patients
infected with Campylobacter spp. than in those infected
with EPEC (p = 0.007, 2-tailed Fisher exact test), aden-
ovirus (p = 0.0005), rotavirus (p = 0.0005), or those in
whom no pathogen was detected (p = 0.003).
The duration of diarrhea was significantly longer in
patients with EPEC than in those infected with adenovirus
(p = 0.002, 2-tailed Student t test), rotavirus (p = 0.0003),
Campylobacter (p = 0.0003), Salmonella (p = 0.02), and
those without an identifiable pathogen (p = 0.02).
Moreover, persistent diarrhea (defined as diarrhea lasting
>14 days) was significantly more common in patients
infected with atypical EPEC than in those infected with
adenovirus, rotavirus, Campylobacter,
those with no pathogen identified (Table 4). Persistent
diarrhea also developed in 4 (36%) of 11 patients infected
with Giardia sp. The frequency of persistent diarrhea asso-
ciated with Giardia was significantly greater than that
attributable to adenovirus (p = 0.03), rotavirus (p = 0.01),
and Camplyobacter, but not Salmonella or atypical EPEC
(p>0.1, 2-tailed Fisher exact test).
Fever was significantly more common in patients infect-
ed with rotavirus or Salmonella than in those infected with
EPEC, adenovirus, or Campylobacter, and those with no
600Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 4, April 2006
pathogen identified. Dehydration of >5% occurred signifi-
cantly more often in patients infected with rotavirus than in
those infected with EPEC, adenovirus, Campylobacter,
Salmonella, and those without an identifiable pathogen.
The disease severity score, determined according to the
criteria of Ruuska and Vesikari (10), was highest in
patients infected with rotavirus followed by Salmonella sp.
The mean severity scores in patients infected with EPEC,
adenovirus, and Campylobacter sp. and those in whom no
pathogen was found were similar. Stools from patients
infected with Campylobacter or Salmonella spp. were
more likely to contain frank blood, although the differ-
ences between patients infected with different etiologic
agents were not significant (p>0.05, 2-tailed Fisher exact
test). Erythrocytes were more commonly detected on
microscopic examination in patients infected with
Campylobacter or Salmonella spp. than in those infected
with EPEC, adenovirus, rotavirus, or no identifiable
pathogen, but the differences were significant with respect
to Campylobacter spp. only. Fecal leukocytes were present
significantly more often in patients infected with
Campylobacter or Salmonella spp. than in those infected
with EPEC, adenovirus, rotavirus, or those with no identi-
The principal aims of this study were to compare the
frequency of atypical EPEC with frequencies of estab-
lished enteropathogens in children attending hospital with
diarrhea and to determine the clinical and laboratory fea-
tures associated with each pathogen. During the first part
of the study (when pathogenic E. coli was sought), atypi-
cal EPEC was the predominant pathogen identified; it was
found in 31% of 134 children compared with 10% for ade-
novirus, 10% for Salmonella sp., 9% for Campylobacter
sp., and 4% for rotavirus. In the second period of the study,
when EPEC was not sought, rotavirus predominated. In
agreement with our findings from a community-based
study in Melbourne and reports from investigators in
Brazil, Norway, and elsewhere (9,20,21), the atypical
EPEC strains obtained in this study were highly heteroge-
neous in terms of O:H serotype and intimin type, which
indicates that the high frequency of atypical EPEC was not
due to an outbreak caused by a limited number of strains.
Also in agreement with our previous study, we observed
that serotypes of EPEC associated with diarrhea differed
from those listed by the World Health Organization as
being characteristic of EPEC (9).
To determine whether atypical EPEC is a cause of diar-
rhea, we compared the clinical and laboratory findings of
children who were infected with these bacteria with those
who were infected with well-established pathogens and
those in whom no pathogens were identified. The hypoth-
esis underlying this investigation was that if atypical EPEC
is not a pathogen, the symptoms, signs, and laboratory
findings in patients infected with these bacteria would be
Atypical Enteropathogenic Escherichia coli Infection
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 4, April 2006 601
the same as those in patients in whom no pathogens were
found. The results showed that diarrhea attributable to
atypical EPEC was significantly more common in boys
and that it persisted significantly longer than diarrhea in
patients without an identifiable pathogen or in those infect-
ed with adenovirus, rotavirus, Campylobacter spp., or
Salmonella sp. This study also showed that infection with
atypical EPEC generally occurred in children <2 years of
age, with 72% <24 months of age compared with 55% for
the first study group as a whole (OR 3.0, 95% CI
1.17–7.85, p = 0.03, 2-tailed Fisher exact test). Infection
with EPEC was associated with vomiting in ≈50% of
patients, was generally not accompanied by fever, abdom-
inal pain, or dehydration, and was not characterized by
fecal blood or leukocytes, indicating that it was not inflam-
matory in nature. The reason for the higher frequency of
atypical EPEC in boys is not known but confirms our
unpublished observations from a community-based study,
in which 55 isolates were obtained from 338 male patients,
and 34 were obtained from 358 female patients (OR 1.85,
95% CI 1.17–2.92, p = 0.009, 2-tailed Fisher exact test).
The validity of the clinical and laboratory assessments
performed in this study was indicated by the confirmation
of the well-known associations of specific pathogens with
particular parameters: younger age of children infected
with EPEC and viruses than those infected with
Campylobacter or Salmonella spp.; rotavirus and
Salmonella infections with fever; rotavirus with dehydra-
tion and an overall greater severity of disease;
Campylobacter sp. with fecal blood; and Campylobacter
and Salmonella spp. with fecal leukocytes (22).
Persistent diarrhea (lasting more then 14 days) eventu-
ally develops in a substantial proportion of children with
acute infectious gastroenteritis and may become chronic,
leading to malabsorption, failure to thrive, and malnutri-
tion (23). Awide range of infectious agents has been impli-
cated in the cause of persistent diarrhea, including viruses,
in particular rotavirus; protozoa, such as Giardia and
Cryptosporidium spp., and bacteria, including E. coli
(23,24). In most cases, however, laboratory investigation
of children with persistent diarrhea fails to yield an identi-
fiable cause. The findings of this study suggest that a num-
ber of these cases may be caused by infection with atypical
EPEC, which is seldom sought in these patients.
Despite the persuasive evidence of a volunteer study
and reports of outbreaks of diarrhea attributed to atypical
EPEC (25,26), the role of atypical EPEC in disease is con-
troversial. In several reports, however, from countries as
diverse as Iran, Norway, Peru, Poland, South Africa, the
United States, and the United Kingdom (20,27–32), as
well as Australia (9,33), atypical EPEC strains have been
identified in children with acute diarrheal disease. Atypical
EPEC has also previously been reported in association
with prolonged diarrhea (34). For example, Hill et al. (35)
reported that of 26 children infected with EPEC requiring
hospital admission for acute diarrhea, life-threatening,
chronic symptoms developed in 6 (23%). Five of these 6
children were infected with EPEC of serogroups O114 or
O128, which frequently do not produce Bfp (14,36).
Notwithstanding these previous reports, however, the cur-
rent study is the first to characterize the illness caused by
atypical EPEC in a systematic way and to compare the fea-
tures of atypical EPEC infection with those of other etio-
logic agents of diarrhea.
The reasons why persistent diarrhea develops more fre-
quently in children infected with atypical EPEC than in
those infected with adenovirus, rotavirus, Campylobacter
or Salmonella spp. are not known. In a recent study,
Mellmann et al. (37) found that only 12 (<9%) of 137
patients who were infected with eae-positive EHEC strains
when investigated within 14 days of the onset of diarrhea
remained culture-positive when retested 3–16 days later,
compared with all 5 patients who were initially infected
with eae-positive, stx-negative E. coli (i.e., atypical EPEC)
(OR 110.4, 95% CI 5.8–2117.6, p<0.0001, 2-tailed Fisher
exact test). These findings indicate that atypical EPEC
may have an innate propensity to persist longer in the
intestine than varieties of E. coli which cause diarrhea that
is more transient in nature. EPEC adheres tightly to epithe-
lial cells and disrupts normal cellular processes (38), and
evidence suggests that atypical EPEC may retard apopto-
sis of intestinal epithelial cells (39), possibly because of
the lack of Bfp (40). These features may favor prolonged
intestinal colonization by atypical EPEC compared with
other intestinal pathogens. Although disease due to atypi-
cal EPEC was mild and generally not associated with
dehydration, its importance lies in its association with pro-
longed diarrhea, a major contributor to childhood illness,
especially in developing countries. Our findings also sug-
gest that interventions targeted towards atypical EPEC
may be beneficial in managing children with prolonged
We are grateful to to K.A. Bettelheim and the staff of the
diagnostic microbiology and virology laboratories at the Royal
Children’s Hospital for their assistance.
This study was supported by grants to R.R.B. from the
Australian National Health and Medical Research Council and
the Murdoch Children’s Research Institute.
Dr Nguyen is head of pediatric infectious diseases at An
Giang General Hospital in Long Xuyen City, An Giang, Vietnam.
His major research interests are viral infections and the develop-
ment of methods for the rapid diagnosis of infectious diseases.
602 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 4, April 2006
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Address for correspondence: R.M. Robins-Browne, Department of
Microbiology and Immunology, University of Melbourne, Melbourne,
Victoria 3010, Australia; fax: 61-3-8344-8276; email: r.browne@
Atypical Enteropathogenic Escherichia coli Infection
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 4, April 2006603