Am. J. Trop. Med. Hyg., 69(5), 2003, pp.

506–508
Copyright © 2003 by The American Society of Tropical Medicine and Hygiene

ASSOCIATION OF VIRULENCE FACTOR-POSITIVE AND -NEGATIVE

ENTEROAGGREGATIVE ESCHERICHIA COLI AND OCCURRENCE OF CLINICAL
ILLNESS IN TRAVELERS FROM THE UNITED STATES TO MEXICO
DAVID B. HUANG, ZHI-DONG JIANG, AND HERBERT L. DUPONT
Department of Medicine, Baylor College of Medicine, Houston, Texas; Center for Infectious Diseases, University of Texas-Houston
School of Public Health and Medical School, Houston, Texas; St. Luke’s Episcopal Hospital, Houston, Texas

Abstract. The objective of the study was to determine if the presence or absence of virulence factor-positive and
-negative enteroaggregative Escherichia coli (EAEC) determined the occurrence of illness or sub-clinical EAEC infec-
tion in travelers from the United States to Mexico. Sixty-five newly arrived college students from the United States
submitted weekly stool samples for a four-week period of time. Among EAEC-infected subjects, diarrhea occurred in
those with a defined virulence factor with the following frequency: aggA, 5 of 15 (33%); aggR, 3 of 11 (27%); aafA, 3
of 8 (38%); and aspU, 1 of 6 (17%). Twenty-two of 31 students (71%) had two or more EAEC infections. After the initial
EAEC infection, only 4 (11%) of 31 students had a subsequent symptomatic EAEC infection. Our study suggests that
clinical illness by EAEC is not explained by presence of a defined EAEC virulence factors, and we provide suggestive
evidence that EAEC infection protects against future symptomatic infection.

INTRODUCTION Diego whose program the students attended in Guadalajara,

Enteroaggregative Escherichia coli (EAEC) has been im-
plicated as a cause of persistent diarrhea in children1 and
acquired immunodeficiency syndrome−associated diarrhea,2,3
as well as acute diarrhea in travelers.4,5 Not all EAEC strains
have been shown to cause diarrhea in humans. The EAEC
are a heterogeneous group of bacteria that display a wide
array of virulence factors. The relative contribution to disease
for each one of these virulence factors is unclear. Further-
more, the interaction between the host immune response and
heterogeneity of virulence factors of EAEC is complex.1,6
This prospective study examined the association of virulence
factor-positive and -negative EAEC with the occurrence of
clinical illness in travelers to Mexico.
MATERIAL AND METHODS
Sixty-five college students from the United States (age
range ⳱ 18−36 years old) attending classes in Guadalajara,
Mexico in July and August 1999 were offered the opportunity
to participate in the study. Inclusion criteria included enroll-
ment in the study and provision of a stool sample for study
within 72 hours of arrival in Mexico and agreement to provide
subsequent stool samples at the end of the first, second, and
third weeks of their time in Guadalajara. Exclusion criteria
included travel to a developing region of the world within
three months or receipt of an antimicrobial agent with ex-
pected activity against bacterial enteric pathogens (e.g., tri-
methoprim-sulfamethoxazole or a fluoroquinolone) within
one week prior to enrollment.
The four stool samples per subject were submitted to our
field laboratory and processed for enteric pathogens. Symp-
tomatic infection or diarrhea was defined as passage of three
or more unformed stools in a 24-hour period plus one or more
signs or symptoms of enteric illness (i.e., nausea, vomiting,
abdominal pain or cramping, fecal urgency, or dysentery).
Participation in this study was voluntary and required the
written informed consent of subjects prior to enrollment. The
study was reviewed and approved by the Committee for the
Protection of Human Subjects of the University of Texas-
Houston Health Science Center and by the University of San
Mexico.
All stool samples were tested for conventional enteric
pathogens as described previously.7
Five E. coli-like colonies per stool sample retrieved from
MacConkey agar plates were transported in individual pep-
tone stabs to Houston. These E. coli-like colonies were then
taken to the Center for Infectious Diseases at the University
of Texas-Houston for further analyses that included detection
of toxin genes of ETEC8 and the HEp-2 cell adherence assay
to detect the patterns of adherence. The presence of EAEC
was determined by the HEp-2 cell adherence assay.9 Briefly,
a chamber slide (Dynatek, Naperville, IL) was seeded with
HEp-2 cells that had been grown at 37°C in 5% CO2 in mini-
mal essential medium (MEM) (Gibco-BRL, Gaithersburg,
MD) supplemented with 10% fetal calf serum. Escherichia
coli to be tested were grown overnight statically at 37°C in
tryptic soy broth (BBL Microbiology Systems, Cockeysville,
MD) with 1% D-mannose. The cell culture medium in the
chamber slide was then replaced with MEM containing 1%
D-mannose without antibiotics and E. coli were added before
incubation at 37°C for three hours. The slide was washed
three times with phosphate-buffered saline, fixed with 100%
methanol, and stained with crystal violet (Difco, Detroit, MI).
Escherichia coli strains H10407, 042, and HS were used
as quality controls. Each E. coli strain was tested twice in a
blinded fashion. A sample was interpreted as positive for
EAEC if it showed the characteristic “stacked-brick” aggre-
gative appearance.9
The EAEC virulence factors were studied in each isolated
EAEC by a PCR. Oligonucleotide primers for EAEC viru-
lence factors aggA, aggR, aafA, and aspU were selected based
on previous studies.1,10 An amplification mix of 98 ␮L of PCR
mixture (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2,
100 ␮M each of dATP, dCTP, dGTP, and dTTP, and 2.5 units
of AmpliTaq polymerase [Perkin-Elmer, Norwalk, CT]), 25
pmol of each primer, and 2 ␮L of DNA template were heated
for five minutes at 94°C. The reaction mixture was then sub-
jected to 35 cycles (94°C for 30 seconds, 50°C for one minute,
and 72°C for 45 seconds) and then to a final extension at 72°C
for seven minutes in a DNA thermal cycler (Perkin-Elmer).
Ten microliters of the amplified PCR product was then sub-

506
 EAEC, VIRULENCE FACTORS, AND CLINICAL ILLNESS
jected to electrophoresis on a 1% agarose gel with a 1-kb
DNA molecular weight marker (Gibco-BRL). A positive re-
sult was determined by the presence of a PCR product of
expected size.
The statistical function used to determine significant differ-
ences was the chi-square test.
RESULTS
Of the 65 students enrolled in this study, 31 (48%) students
had at least one stool sample positive for an EAEC during the
four-week study. Sixty-five stool samples were provided at
week one, 62 at week two, 59 at week three, and 55 at week
four. In the 31 students who had at least one EAEC isolated
from their stools, 31 stool samples were provided at week one,
30 at week two, 28 at week three, and 25 at week four. Eigh-
teen (58%) of the 31 students had an asymptomatic infection
and 13 (42%) students had a symptomatic EAEC infection.
Including both asymptomatic and symptomatic infections,
22 (71%) of 31 students had two or more EAEC infections.
Nine students (29%) had one sample positive for EAEC, 13
(42%) students had two, six (19%) had three, and three
(10%) had four weekly stool samples positive for EAEC.
The number of symptomatic EAEC infection by virulence
factor and by week of study is shown in Table 1. AggA
(37.5%) and aggR (27.5%) were the two most commonly iso-
lated virulence factors. Fifteen EAEC isolates had the viru-
lence factor aggA. Of those 15, five (33.3%) had a symptom-
atic EAEC infection. Eleven (27.5%) students had an EAEC
isolated with the virulence factor aggR, and three (27.3%) of
those 11 students developed diarrheal illness. Eight (20%)
students had an EAEC isolated positive for the virulence
factor aafA and three experienced diarrhea (37.5%). Six
(15%) of the students were positive with the virulence factor
aspU and one (16.7%) developed diarrhea.
The number of initial and subsequent EAEC infection in
the various students is shown in Figure 1. Nine (29%) of the
31 students had an initial symptomatic infection, and 22
(71%) of the students had an initial asymptomatic EAEC
infection. After the initial EAEC infection, four (13%) stu-
dents had a subsequent symptomatic EAEC illness (P ⳱ 0.12,
95% confidence interval ⳱ 0.78−9.62), and 27 students (87%)
developed asymptomatic EAEC reinfection.
DISCUSSION
The present study failed to show an association between
virulence factor-positive EAEC and the occurrence of clinical
illness in travelers from the United States to Mexico. Viru-
TABLE 1
Number symptomatic/total enteroaggregative Escherichia coli infec-
tions by virulence factor and week of study in United States stu-
dents recently arrived in Mexico
Week of study after arrival in Mexico
Virulence
factors 1 2 3 4
aggA 1/3 (33.3) 2/3 (66.7) 0/5 2/4 (50)
aggR 0/1 2/2 (100) 0/5 1/3 (33.3)
aspU 0/1 0 0/4 1/1 (100)
aafA 0/1 2/2 (100) 0/2 1/3 (33.3)
507
FIGURE 1. Students from the United States in Mexico with initial
and subsequent culture-proven enteroaggregative Escherichia coli
(EAEC) symptomatic and asymptomatic infections. 1 ⳱ Initial symp-
tomatic EAEC infections; 2 ⳱ Initial asymptomatic EAEC infec-
tions; 3 ⳱ Subsequent symptomatic EAEC infections; 4 ⳱ Subse-
quent asymptomatic EAEC infections. Values above each bar are the
no. (%).
lence factor-positive EAEC occurred in asymptomatic stu-
dents, and virulence factor-negative EAEC occurred in symp-
tomatic students. One student had an asymptomatic EAEC
infection that carried all four virulence factors: aggA, aggR,
aafA, and aspU. These results suggest that host genetic and
environmental factors may be more important in influencing
the disease outcome of EAEC infection. Also, additional
virulence properties of EAEC strains may help to explain
human virulence for this emerging pathogen.
The most common virulence factor isolated from EAEC
was aggA (37.5%). AggR and aafA were commonly found,
seen in 27.5% and 20% of EAEC strains isolated, respec-
tively. AspU was the least commonly found virulence factor,
found in 15% of tested strains. Other studies have also shown
that aggA and aggR are common EAEC virulence factors.11
Most EAEC strains possess a 60−65-mD plasmid, designated
pAA (aggregative adherence), which encodes several viru-
lence factors such as the AA fimbria, AAF/I, and AAF/II.10
The AAF/I genes include aggA, which encodes the major
fimbrial subunit. The AAF/II genes include aafA, which has
been shown to mediate adherence to the intestinal mucosa.12
Both AAF/I and AAF/II require the action of the transcrip-
tional activator aggR. AspU is a cryptic gene that encodes for
a secreted protein. The precise clinical relevance of any of
these virulence factors remains unclear.
Seventy-one percent of the students developed two or more
EAEC infections during their four weeks stay in Mexico. In a
previous study using the same student population, a plasmid
DNA analysis revealed a high degree of heterogeneity among
the infecting EAEC isolates.13 The presence of multiple
Total strains circulating in the population has also been shown by
5/15 (33.3) the different virulence properties that they possess, as well as
3/11 (27.3) by their pulsed-field gel electrophoresis, and plasmid DNA
1/6 (16.7) content.5,6,10,13
3/8 (37.5)
Although not statistically significant, our data suggest that
508 HUANG AND OTHERS
early symptomatic or asymptomatic infection with EAEC is
associated with protection against subsequent symptomatic
EAEC infection. Twenty-two (71%) of 31 EAEC infections
occurred one or two weeks after arrival in Mexico. Regardless
of the presence of symptomatic or asymptomatic EAEC in-
fection, there was an 85−91% chance of not developing sub-
sequent EAEC diarrhea once infection had occurred. These
results suggest that the early EAEC infection “primes” the
immune system of the host by providing protection against
future symptomatic EAEC infections regardless of presence
of one of virulence properties studied. Immunity does not
appear to develop to asymptomatic EAEC infection because
many of the students developed multiple EAEC infections
with isolated strains possessing different virulence factors. No
fecal IgA was measured in this study. However, it is likely that
this immunoglobulin is involved in the protection of subse-
quent EAEC infection. This is an area of future interest in our
laboratory.
All EAEC isolates were identified by the HEp-2 cell as-
say.9 The HEp-2 assay remains the “gold standard” for iden-
tification of EAEC. We believe that this assay is accurate and
that presence of bacterial clusters in a stacked-brick configu-
ration provides definitive identification of EAEC strains. No
analysis of the pAA plasmid was performed in this study. It
may, however, be of interest to determine the genotype cor-
relate associated with phenotype. Other diagnostic tools of
EAEC are currently being evaluated. A DNA probe based on
a 1.0-kb DNA fragment labeled PCVD432 from the 60-mD
plasmid of EAEC strain 17-2 has been shown to have varying
results with a sensitivity of 15−89% and a specificity of 99%
compared with the HEp-2 cell assay.14–16 These varying re-
sults are likely due to the assumption that the DNA se-
quences targeted correspond to a conserved virulence factor.
However, EAEC is a heterogeneous pathogen, and many
EAEC strains lack definable virulence properties making ge-
notype correlates with phenotype difficult.
An additional area of interest is the possibility of plasmid
loss upon transport. It is a largely unstudied area, and there is
no existing evidence that loss occurs with EAEC.
In conclusion, there was no association of presence or ab-
sence of one or more of four defined virulence factors in
EAEC infection and the occurrence of clinical illness. Many
students had multiple EAEC infections during their four-
week stay in Mexico. Early infection with EAEC appears to
be associated with protection against subsequent symptom-
atic EAEC infection. Our study suggests that the spectrum of
disease is likely determined by both the presence of patho-
gen-specific virulence factors and by the ability of the host to
respond to the inflammatory stimulus caused by EAEC.
Received June 16, 2003. Accepted for publication August 20, 2003.
Authors’ addresses: David B. Huang, Department of Medicine, Bay-
lor College of Medicine, 1 Baylor Plaza, BCM 286, Room N1319,
Houston, TX 77030, Telephone: 832-355-4122, Fax: 832-355-4167, E-
mail: dhuang1@bcm.tmc.edu. Zhi-Dong Jiang, University of Texas
School of Public Health, 1200 Herman Pressler, Room 706, Houston
TX 77030 and University of Texas-Houston Medical School, 1200
Herman Pressler, Room W703, Houston, TX 77030, E-mail:
zjiang@sph.uth.tmc.edu. Herbert L. DuPont, St. Luke’s Episcopal
Hospital, 6720 Bertner Avenue, MC -1-164, Houston, TX 77030, E-
mail: hdupont@sleh.com.
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