International Journal of Infectious Diseases 15 (2011) e136–e139

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International Journal of Infectious Diseases

journal homepage: www.elsevier.com/locate/ijid

Characterization of enteroaggregative Escherichia coli (EAEC) clinical isolates

and their antibiotic resistance pattern
Mohammad Mehdi Aslani a, Mohammad Yousef Alikhani b,*, Ali Zavari b, Rasol Yousefi b, Ali Reza Zamani c
a
Microbiology Department, Pasteur Institute of Iran, Tehran, Iran
b
Microbiology Department, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
c
Immunology Department, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran

A R T I C L E I N F O S U M M A R Y

Article history: Objectives: Enteroaggregative Escherichia coli (EAEC) strains are an emerging type of diarrheagenic E. coli.
Received 21 February 2010 The aim of this study was to investigate the frequency of EAEC in children with diarrhea by polymerase
Received in revised form 25 September 2010 chain reaction (PCR) method targeting the pCVD432 gene. The presence of virulence genes including
Accepted 4 October 2010
aggR, aggA, aafA, aap, and astA was also investigated by PCR, for the differentiation of typical and atypical
Corresponding Editor: EAEC strains. We also sought to determine the antibiotic resistance pattern of the isolated strains.
Karamchand Ramotar, Methods: Stool samples were collected from 140 children with diarrhea at Besat Hospital, Hamadan,
Ottawa, Canada
Iran, from July 2007 to May 2008. The specimens were cultured for E. coli, which was identified using
standard methods. E. coli strains were screened for EAEC by PCR and HeLa cell line adherence methods.
Keywords: For each sample, five single colonies (700 E. coli strains) from original MacConkey plates were examined
Enteroaggregative Escherichia coli for pCVD432, aggR, aggA, aafA, aap, and astA genes using PCR. The EAEC adherence patterns were
EAEC
examined by HeLa cell adherence method. Antimicrobial susceptibility testing was performed as the
HeLa cell
Clinical and Laboratory Standards Institute (CLSI) guidelines.
Antibiotic resistance
Results: Overall, 15 (10.7%) EAEC strains were identified in 140 diarrhea cases by PCR. Out of these
isolates, EAEC were detected in 13 (86.7%) by the HeLa cell assay. The aggR regulon was present in 11
(73.3%) strains. Several different combinations of the virulence markers were found among the EAEC
isolates. The most prevalent (20%) combination was aggR–aap–astA. The EAEC isolates exhibited
resistance to ampicillin (100%), erythromycin (100%), cephalothin (78.6%), co-trimoxazole (71.4%),
tetracycline (64.3%), and nalidixic acid (57.1%) and reduced resistance to ciprofloxacin (42.9%) and
norfloxacin (7.1%).
Conclusions: EAEC is a diarrheal pathogen of emerging importance. Correlation between pCVD432 PCR
and the HeLa cell line assay was confirmed in children with diarrhea. In comparison to the assay for
aggregative adherence, the EAEC PCR has been found to be simple and specific in many epidemiological
studies. The typical EAEC (73.3%) strains (with pCVD432 and aggR genes) identified in this study were
heterogeneous with respect to virulence genes. This study also showed that EAEC isolates were highly
resistant to tetracycline, co-trimoxazole, and ampicillin, which are the most commonly used antibiotics
in our area.
ß 2010 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

chain reaction (PCR) method based on identifying the presence of

1. Introduction
Enteroaggregative Escherichia coli (EAEC) strains are causal
agents of persistent diarrhea in developing countries.1,2 EAEC
strains are determined by their characteristic aggregative adher-
ence (AA) pattern to HEP-2 or HeLa cell lines in a stacked brick-like
pattern.3 However, the adhesion method is not suitable as a
diagnostic assay since it is difficult to perform and the results have
to be interpreted by experienced microbiologists.4 A polymerase
* Corresponding author. Tel.: +98 811 8276294; fax: +98 811 8276299.
E-mail address: alikhani@umsha.ac.ir (M.Y. Alikhani).
specific EAEC genes may provide a more efficient way to diagnose
EAEC strains in fecal samples.
The AA phenotype in EAEC strains is associated with the
presence of a 60-MDa plasmid (the AA plasmid),5,6 which codes
many virulence genes, including an anti-aggregation protein
transporter (CVD432 or the AA probe),7,8 enteroaggregative heat
stable toxin (EAST; astA),9,10 aggregative adherence fimbria I (AAF/
I; aggA), aggregative adherence fimbria II (AAF/II; aafA), dispersin
secretory protein (aap), and the gene encoding transcriptional
activator AggR (aggR), which is needed for expression of fimbriae.11
EAEC strains are a heterogeneous group of E. coli with regard to the
symptoms that develop in infected hosts and the virulence genes.

1201-9712/$36.00 – see front matter ß 2010 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijid.2010.10.002
 M.M. Aslani et al. / International Journal of Infectious Diseases 15 (2011) e136–e139
Resistance to antibiotics is very common in bacterial isolates
worldwide. Regular surveillance of antibiotic resistance provides
information for antibiotic therapy and resistance control.
The objectives of this study were (1) to determine the prevalence
of EAEC by PCR and HeLa cell adhesion assays among Iranian patients
with diarrhea, (2) to determine the antimicrobial resistance profiles
of EAEC strains, and (3) to determine the prevalence of genes for
virulence factors associated with EAEC strains.
2. Methods
2.1. Fecal samples
Between July 2007 and May 2008, 140 fecal samples were
collected from children aged <12 years with diarrhea attending
Besat Hospital, affiliated to Hamadan University of Medical Sciences,
Hamadan, Iran. Of the 140 patients included in the study, 83 were
male and 57 female. The specimens were cultured for E. coli,
Salmonella spp, Shigella spp, Vibrio cholerae, and Campylobacter spp
using standard methods. E. coli strains were then screened for EAEC
using PCR and the HeLa cell line adhesion assay.
2.2. EAEC identification by PCR
The fecal samples were plated onto MacConkey agar and
incubated overnight at 37 8C. A nutrient broth tube was inoculated
with a sweep of mixed colonies from the MacConkey plates and
incubated at 37 8C for 4 h. Template DNA was prepared from bacteria
grown in nutrient broth by boiling method for use in PCR. This
culture was centrifuged to pellet the bacteria, the supernatant was
removed, and the pellet was resuspended in 300 ml sterile distilled
water and heated for 10 min at 100 8C. Samples were recentrifuged
at 14 000 g. The supernatants were collected in sterile tubes and
stored at 20 8C. The samples were examined for the pCVD432
gene12 by PCR (the primers used are shown in Table 1). In samples of
mixed colonies with a positive PCR result, a pure-colony pick
containing pCVD432 was obtained from the original MacConkey
plate in all cases as follows. Five single colonies from each plate (700
E. coli strains) were inoculated into nutrient broth and incubated at
37 8C for 4–6 h. The single colonies were examined for the pCVD432
gene using PCR, as described above. The single colonies positive for
pCVD432 were examined for aggR,13 aggA,14 aafA,15 aap,16 and astA17
genes, as described previously (Table 1).
EAEC strain 17-26 and E. coli K12 (ATCC 10798) were used as
positive and negative control, respectively.
2.3. HeLa cell line adhesion assay
All pCVD432-positive isolates were examined for HeLa cell
adherence as described by Scaletsky et al.,18 with slight modifica-
Table 1
Primers used in this study
Target gene Primer sequence (50 to 30 ) Size of product (bp) Ref.
0 0
pCVD432 F-5 -CTGGCGAAAGACTGTATCAT-3 630
R-50 -AATGTATAGAAATCCGCTGTT-30
aggR F-50 -GTATACACAAAAGAAGGAAGC-30 254
R-50 -ACAGAATCGTCAGCATCAGC-30
aggA F-50 -TTAGTCTTCTATCTAGGG-30 457
R-50 -AAATTAATTCCGGCATGG-30
aafA F-50 -TGCGATTGCTACTTTATTAT-30 242
R-50 -ATTGACCGTGATTGGCTTCC-30
aap F-50 -CTTGGGTATCAGCCTGAATG-30 310
R-50 -AACCCATTCGGTTAGAGCAC-30
astA F-50 -CCATCAACACAGTATATCCGA-30 111
R-50 -GGTCGCGAGTGACGGCTTTGT-30
e137
tions. Briefly, monolayer HeLa cells (National Cell Bank of Iran,
Pasteur Institute of Iran) were grown to 50% confluency on cover
slips in Leighton tubes in the presence of 1% mannose and infected
with bacteria for 3 h at 37 8C in 5% CO2. The infected monolayers
were washed with sterile phosphate-buffered saline, fixed with
70% methanol, stained with 10% Giemsa stain, and examined for AA
pattern under a light microscope using a 40 objective. Prototype
EAEC strain 17-2 (serotype O3:H2)6 was used as positive control
and E. coli K12 (ATCC 10798) as negative control.
2.4. Antibiotic resistance tests
The strains were tested for antibiotic resistance by the disk
diffusion method according to the Clinical and Laboratory Standards
Institute (CLSI; formerly NCCLS) guidelines.19 The following
antibiotic disks were used: ampicillin, erythromycin, cephalothin,
co-trimoxazole, tetracycline, nalidixic acid, co-amoxiclav, cefotax-
ime, ceftriaxone, chloramphenicol, gentamicin, ciprofloxacin, nor-
floxacin, and cefixime (HiMedia Laboratories Co., India).
3. Results
Of the 140 patients included in the study, 83 (59.3%) were male
and 57 (40.7%) female. Eighty-two samples were from children
younger than 5 years of age, and 58 were from children aged
between 5 and 10 years. Examining the seasonal occurrence of
diarrhea from all causes showed that gastroenteritis was more
common in the warmer months; 79.3% of cases occurred from June
through November. Infections with EAEC reflected this distribu-
tion, with 80% of infections caused by these bacteria occurring
during the same period.
Strains of E. coli harboring the pCVD432 gene were found as the
sole pathogen in the fecal samples of 15 (10.7%) children with
diarrhea; no mixed infections were observed. Twelve (80%)
children with E. coli positive for the pCVD432 gene were under
5 years old. All EAEC isolates were tested by PCR to detect the genes
of the proposed EAEC virulence factors, including aggR, aggA, aafA,
aap, and astA. Out of the 15 patients who had isolates harboring the
pCVD432 gene, 11 (73.3%) also had the aggR gene; these were
classified as typical EAEC (tEAEC). The aap gene was found in nine
isolates, and seven strains had the astA gene. Strains of E. coli
harboring only the aggR gene, but not the other virulence genes,
were isolated from two patients (13.3%), and showed an
aggregative phenotype. Of the 15 patients who had isolates
harboring the pCVD432 gene, four also had the aafA gene, three
carried the aggA gene, and 13 showed AA to HeLa cells in a stacked-
brick formation. Three patients had strains of E. coli that carried the
aggR, aap, and astA genes, but not the aafA and aggA genes, and
showed an AA pattern (Table 2). Only one pCVD432-positive strain
was non-adherent and lacked all the other virulence factor genes.
Several different combinations of the virulence markers were
found among the EAEC isolates (Table 3). The most prevalent
combination was aggR, aap, and astA, found in three (20%) strains.
The results of antimicrobial susceptibility testing of the
diarrheagenic EAEC isolates to 14 antibiotics by disk diffusion
method are shown in Table 4. Of the 14 tested strains, 10 (71.4%)
12
were multidrug-resistant (resistance to more than three antimi-
13 crobial drugs). The most prevalent resistance profile was ampicil-
lin–erythromycin–cephalothin and co-trimoxazole.
14
4. Discussion
15
16 Enteroaggregative E. coli (EAEC) form a subgroup of diarrhea-
genic E. coli (DEC) that has received increasing attention during the
17 past decade as a cause of watery diarrhea, which is often
persistent.20,21 EAEC have been isolated from sporadic cases and
e138 M.M. Aslani et al. / International Journal of Infectious Diseases 15 (2011) e136–e139

Table 2 Table 4
Characterization of the adhesion and virulence factors of EAEC strains isolated from Antimicrobial susceptibility of EAEC isolates from children with diarrhea
diarrhea patients
Antimicrobial agent Resistant, n (%) Susceptible, n (%) Intermediate, n (%)
Isolate Virulence factors HeLa adhesion
Ampicillin 14 (100) 0 (0) 0 (0)
aggR aggA aafA aap astA Erythromycin 14 (100) 0 (0) 0 (0)
Cephalothin 11 (78.6) 1 (7.1) 2 (14.3)
EAEC1 +   + + AA
Co-trimoxazole 10 (71.4) 3 (21.5) 1 (7.1)
EAEC2 + +  +  AA
Tetracycline 9 (64.3) 0 (0) 5 (35.7)
EAEC3      NA
Nalidixic acid 8 (57.1) 0 (0) 6 (42.9)
EAEC4 +   + + AA
Cefixime 7 (50) 1 (7.1) 6 (42.9)
EAEC5      AA
Co-amoxiclav 7 (50) 0 (0) 7 (50)
EAEC6    +  AA
Cefotaxime 6 (42.9) 2 (14.2) 6 (42.9)
EAEC7 +     AA
Ceftriaxone 6 (42.9) 2 (14.2) 6 (42.9)
EAEC8 +   + + AA
Chloramphenicol 5 (35.7) 7 (50) 2 (14.3)
EAEC9     + NA
Gentamicin 4 (28.6) 4 (28.6) 6 (42.9)
EAEC10 +     AA
Ciprofloxacin 0 (0) 8 (57.1) 6 (42.9)
EAEC11 +   +  AA
Norfloxacin 0 (0) 13 (92.9) 1 (7.1)
EAEC12 + + +  + AA
EAEC13 + + + + + AA EAEC, enteroaggregative Escherichia coli.
EAEC14 +  + + + AA
EAEC15 +  + +  AA
No. (%) 11 (73.3) 3 (20) 4 (26.7) 9 (60) 7 (46.7)

EAEC, enteroaggregative Escherichia coli; AA, aggregative adherence; NA, non-
adherence.
outbreaks worldwide, affecting children and adults.20,21,22 Our
study also shows EAEC as a cause of sporadic diarrhea.
In this study, 140 children with diarrhea were examined for
EAEC. A total of 15 EAEC were isolated from fecal specimens as sole
pathogens; 80% of the EAEC were isolated from children under 5
years old. The PCR results for the pCVD432 gene showed good
correlation with the HeLa cell adhesion assay, as 86.7% of
pCVD432-positive isolates were confirmed as EAEC. Baudry
et al.7 first used the pCVD432 gene as a target for the identification
of EAEC, and declared that the assay was 89% sensitive. However,
subsequent studies have shown that the sensitivity varies between
15% and 90%.23 In our study the pCVD432 PCR in comparison with
the HeLa cell culture assay showed 100% sensitivity, 98.4%
specificity, 86.7% positive predictive value, and 100% negative
predictive value. The pCVD432 gene has remained the most
common target in molecular assays for the detection of
EAEC.12,16,22,24 Epidemiological and pathogenetic studies have
suggested that pCVD432 gene-positive strains that carry the AggR
regulon may be the tEAEC pathogens.25 The tEAEC (73.3%) strains
identified in this study were heterogeneous with respect
to virulence genes. Previous studies have shown that DEC strains
are among the most important agents of diarrhea in Iran.26,27,28
Our results agree with these studies and show that in this area,
EAEC strains, especially tEAEC strains, are one of the enteric
pathogens in children.
Several different combinations of the virulence markers were
found among the EAEC isolates in our study (Table 3). In the past,
Table 3
The prevalence of different virulence genes among EAEC strains isolated from
children with diarrhea
Genetic profile
No virulence factor
aap
astA
aggR
aggR, aap
aggR, aap, aggA
aggR, aap, aafA
aggR, aap, astA
aggR, aap, aafA, astA
aggR, aggA, aafA, astA
aggR, aap, aggA, aafA, astA
EAEC, enteroaggregative Escherichia coli.
the astA gene was considered characteristic of EAEC strains,9
although it has since been detected in only a subgroup of EAEC and
has an extensive distribution among other pathogenic and non-
pathogenic E. coli strains.29,30 In our study, seven (46.7%) of the
EAEC isolates harbored the astA gene and six of them showed an
aggregative adherence pattern.
Other studies have shown that the prevalence of astA-positive E.
coli in the stool specimens of adults and children without diarrhea
can be as high as 38%.29,31 However, some E. coli strains isolated
from people with diarrhea have been shown to harbor no virulence
factors other than EAST.32,33 This PCR assay detected a variety of
strains, demonstrating the traits of EAEC strains, and showing the
usefulness of this method for identifying both typical and atypical
EAEC.
Antimicrobial susceptibility testing results for the EAEC strains
are shown in Table 4. The high incidence of antibiotic-resistant
isolates of EAEC may be due to the widespread use of antibiotics.
The high levels of quinolone-resistant and intermediate isolates
may be the result of the high level of prescription of these drugs in
our area. The repeated use of even a single antibiotic for weeks or
months will lead to the selection of bacteria resistant to the
antibiotic. The transfer of resistance genes that may occur between
species could lead to the construction of diverse resistance to the
usual antibiotics, and resistance to antibiotics can be increased
from the native variant.34,35
Sang et al.36 described four cases of diarrhea caused by multi-
resistant EAEC strains in Kenyan children and concluded that these
multi-resistant DEC strains might also appear in other developing
countries where the classical antibiotics ampicillin, tetracycline,
and trimethoprim–sulfamethoxazole are widely used. The fre-
quencies of antimicrobial resistance have been shown to be high in
EAEC strains in our country.37 In other studies, EAEC have been
found to be resistant to more antibiotics than other DEC
pathotypes.38 Ampicillin and co-trimoxazole are recommended
by the World Health Organization for the treatment of severe or
No. (%) of EAEC persistent diarrhea. However, local information on antimicrobial
resistance patterns should be used in clinical management, and
2 (13.3)
1 (6.7)
treatment procedures should be updated accordingly.39 On the
1 (6.7) basis of local information, ampicillin and co-trimoxazole should
2 (13.3) not be used in our area. One alternative for the treatment of
1 (6.7) diarrhea is the use of quinolones. However, fluoroquinolones are
1 (6.7)
not recommended for children. Moreover, it should be taken into
1 (6.7)
3 (20) account that despite the minimal use of fluoroquinolones in
1 (6.7) children, we detected nalidixic acid-resistant E. coli strains (57.1%).
1 (6.7) Where the use of antibiotics is not controlled, the use of quinolones
1 (6.7) as a first-line therapy for enteritis will result in dissemination of
quinolone resistance. However, considering the high level of
 M.M. Aslani et al. / International Journal of Infectious Diseases 15 (2011) e136–e139
resistance to antibiotics among EAEC, it appears that supportive
treatment including rehydration therapy, and not antimicrobial
therapy, may be the most effective method for the management of
diarrhea caused by EAEC.
Funding
The funding source for our research was Hamadan University of
Medical Sciences, Hamadan, Iran.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
We are grateful to Dr A. Jafari for critical discussions and to Miss
Z. Haidarbarghi for her technical help. Our thanks also go to the
authorities and health care personnel of Besat Hospital, Hamadan,
for the collection of samples.
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