Abstract
We have previously shown that enteroaggregative Escherichia coli (EAEC) is an important pathogen among Brazilian infants. Most EAEC strains harbor a plasmid (pAA) from which a DNA fragment has been used as a probe (EAEC probe). To better understand the characteristics of EAEC in Brazil, 109 strains carrying and lacking the EAEC probe sequence were tested for the presence of pAA plasmid-borne and chromosomal factors. Common virulence factors of probe-positive and probe-negative isolates included the presence of the Pet, EAST-1, Shf, Irp2, ShET1/Pic, and Hly virulence markers. The presence of AggR or one other virulence factor (AAF/I, AAF/II, AAF/III, or Aap) was predominantly identified only in probe-positive strains. In EAEC probe-positive strains, the virulence marker Aap was found significantly more frequently (P = 0.023) in isolates from children with diarrhea (22%) than in isolates from controls (3%). EAST-1 and Shf were the markers most frequently detected (61%) in EAEC probe-negative strains and were found to be significantly associated with diarrhea (P = 0.003 and P = 0.020, respectively). Furthermore, our data suggest that AggR can be used as an important genetic marker for EAEC probe-positive strains.
Enteroaggregative Escherichia coli (EAEC) is increasingly recognized as a cause of diarrhea worldwide (18). EAEC is defined by its characteristic “stacked brick” aggregative adherence (AA) pattern of adherence to HEp-2 cell (17). Most EAEC strains harbor a 60- to 65-MDa virulence plasmid (pAA). A 1-kb fragment of pAA, referred to as the EAEC probe or CVD432 (1), has been widely used for epidemiological studies (7, 19, 25, 29). The pAA plasmid also encodes AA fimbriae (AAF) I, II, and III (2, 4, 15); the transcriptional activator AggR (16); enteroaggregative heat-stable enterotoxin 1 (EAST-1) (23); a 104-kDa cytotoxin designated Pet (8); the cryptic secreted protein Shf (5); and a novel antiaggregation protein (dispersin) encoded by the aap gene (formerly known as aspU) (27). In addition to the pAA plasmid, some EAEC strains express putative virulence factors that are encoded on the chromosome, including a 116-kDa secreted mucinase (Pic) (12), yersiniabactin (26), and the E. coli α-hemolysin (30). Shigella enterotoxin 1 (ShET1) is encoded by the antisense strand of the pic gene (10, 21). However, none of these factors is consistently found in all EAEC isolates, as determined by hybridization studies.
To find a specific virulence marker for the detection of EAEC in epidemiological and clinical studies, we tested 109 strains, isolated from Brazilian children in a previously described study (25), for their abilities to hybridize to eight pAA plasmid-derived and three chromosomal gene probes.
MATERIALS AND METHODS
Bacterial strains.
The strains examined in this work were isolated during a study of the epidemiology of acute diarrhea in children less than 2 years old conducted in different regions of Brazil from 1997 to 1999 (25). The children were admitted to public hospitals in the following cities (states) for treatment: São Paulo (São Paulo), Joinville (Santa Catarina), Natal (Rio Grande do Norte), Goiania (Goiás), and São Luiz (Maranhão). That study used rectal swab specimens from 338 children with acute diarrhea (case patients) and 322 asymptomatic children, matched for age with the case patients, without any gastrointestinal symptoms for at least 30 days prior to inclusion in the study (controls).
In the study mentioned above, each fecal specimen was examined by standard methods for the presence of Shigella spp., Salmonella spp., Giardia lamblia, Yersinia enterocolitica, Campylobacter spp., Cryptosporidium spp., and rotavirus. Four separate lactose-fermenting colonies and two non-lactose-fermenting colonies of each distinct morphological type were cultivated in commercial test systems (PROBAC do Brasil, São Paulo, Brazil), for biochemical confirmation of the species or genus. All E. coli isolates were tested with specific DNA probes designed to detect enterotoxigenic E. coli (heat-labile toxin- and heat-stable toxin-specific probes), enteroinvasive E. coli (Inv-specific probe), Shiga-toxin-producing E. coli (Shiga toxin 1- and Shiga toxin 2-specific probes), EAEC (EAEC-specific probe), diffusely adhering E. coli (daaC- and AIDA-I-specific probes), and EPEC (eae- and EAF-specific probes).
Serotyping.
Identification of the somatic (O) antigens of the strains was done by standard agglutination methods (9) with specific O1 to O175 antisera acquired commercially (Universidad de Santiago de Compostela, Santiago de Compostela, Spain). Twelve strains were tested in the Enteric Section of the Instituto Adolfo Lutz (São Paulo, Brazil) by using H antisera prepared with type strains.
DNA hybridization.
Hybridization assays were performed with specific DNA fragments amplified from prototype strains or fragments derived from cloned DNA probes (Table 1). These fragments were labeled with [α-32P]dCTP, and colony hybridization assays were performed as described previously (25).
TABLE 1.
Probes hybridizing to colony blots of EAEC isolates
| Target gene | Properties of target | Gene location | Fragment (reference) |
|---|---|---|---|
| aggA | AAF/I subunit | Plasmid | 450-bp amplified fragment (24) |
| aafA | AAF/II subunit | Plasmid | 550-bp amplified fragment (4) |
| agg3A | AAF/III subunit | Plasmid | 462-bp amplified fragment (2) |
| astA | EAST-1 toxin | Plasmid | 111-bp amplified fragment (31) |
| pet | 104-kDa cytotoxin | Plasmid | 832-bp fragment of pCEFN1 (8) |
| aap | 10-kDa secreted protein | Plasmid | 232-bp amplified fragment (5) |
| aggR | Transcription of AAFs | Plasmid | 308-bp amplified fragment (5) |
| shf | Cryptic ORFa | Plasmid | 613-bp amplified fragment (5) |
| shET1/pic | Shigella enterotoxin 1/mucinase | Chromosome | 1,100-bp amplified fragment (19) |
| irp2 | Yersiniabactin | Chromosome | 264-bp amplified fragment (5) |
| hly | α-Hemolysin | Chromosome | 6,400-bp fragment of pSF4000 (30) |
ORF, open reading frame.
Statistical analysis.
Data for children with diarrhea and for the controls were compared by a two-tailed chi-square or Fisher's exact test.
RESULTS
A total of 338 children with diarrhea and 322 matched controls without diarrhea were studied. We identified potential diarrheagenic E. coli strains by HEp-2 cell adherence assays and by their abilities to hybridize with specific DNA probes. Of the 660 fecal specimens analyzed, 109 (67 from patients and 42 from controls) were identified as EAEC strains by the AA pattern. Eight-one (74%) of the 109 EAEC strains hybridized with the EAEC-specific probe. Fourteen EAEC strains hybridized with the daaC-specific probe for diffusely adherent E. coli and none of the 109 strains reacted with the probes for enteropathogenic E. coli (EPEC). The number of E. coli isolates with these characteristics in each specimen varied from one to three. For the present study, only one isolate was selected from each of these 109 specimens. The isolates in only 14 of the 109 specimens (8 from patients and 6 from controls) were EPEC, diffusely adherent E. coli, Shigella spp., or rotavirus. In our study, although EAEC was not significantly associated with diarrhea, EAEC isolates were recovered more frequently from children with diarrhea than from children without diarrhea.
In total, 81 EAEC probe-positive and 28 EAEC probe-negative strains were characterized by DNA hybridization to detect the genes for the proposed EAEC virulence factors. The frequencies of strains with different genetic profiles regarding their association with diarrhea were further analyzed.
Characteristics of EAEC probe-positive strains.
Of the 81 EAEC probe-positive strains, 73 (90%) strains belonged to 34 distinct serogroups, of which O44 and O49 were the most frequent (Tables 2 and 3). The virulence factor aggR was the single most common gene marker identified and was found in 43 strains (88%) from patients and 31 strains (97%) from controls. All strains hybridized with three or more pAA-derived probes, with three being the modal value. Strains hybridizing with at least one of the probes for AAF/II, Aap, and Pet were more commonly recovered from children with diarrhea than from controls. The astA, shf, shET1/pic, and hly virulence markers were detected at similar frequencies from patients and controls. The irp2 gene was found more frequently in controls than in patients. When we investigated the strains for the presence of combinations of genes, we found that 48 EAEC strains (59%) possessed aggR and irp2, followed by 36 EAEC strains (44%) possessing the combination aggR and shf; 3 strains were shf positive, and 1 strain was shf and irp2 positive but aggR negative.
TABLE 2.
Distributions of plasmid and chromosomal genes among 49 EAEC probe-positive isolates from patients
| Strain | Serotype or serogroupb | Probe result
|
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Plasmid gene
|
Chromosomal gene
|
|||||||||||
| AAF/I | AAF/II | AAF/III | aggR | aap | astA | pet | shf | shET1/pic | irp2 | hly | ||
| RN785-1 | O1 | − | − | + | + | + | − | − | − | + | + | − |
| HSP161-1 | O3:H2 | − | − | + | + | − | − | − | + | − | + | − |
| MA817-1 | O5 | + | − | − | + | + | + | + | + | − | + | − |
| MA555-7 | O17:NM | − | − | + | + | − | + | + | + | − | + | − |
| RN781-7 | O17 | − | − | − | + | + | − | − | − | − | − | + |
| MA687-3 | O18:NM | − | − | + | + | − | + | − | + | − | + | − |
| MA563-2 | O21 | − | − | − | − | − | − | − | + | − | − | − |
| RN463-2 | O38 | − | − | − | − | − | − | − | + | − | − | − |
| SC725-1a | O40 | − | + | − | + | + | + | + | + | − | + | − |
| HDV139-1a | O42:H28 | − | + | − | + | − | + | + | + | − | − | − |
| HDV147-6 | O42 | − | − | + | + | − | − | + | − | − | + | − |
| HSP41-1 | O44:H18 | − | + | − | + | + | + | + | + | − | + | − |
| MA233-1 | O44 | − | − | + | + | − | − | − | − | − | + | + |
| RN459-1a | O44:NM | − | + | − | + | − | + | − | + | − | + | − |
| SC717-4 | O44 | − | − | − | + | − | − | − | − | − | + | − |
| SP21-2 | O48:H18 | − | − | − | − | − | − | − | − | − | − | − |
| HDV73-1 | O49:H18 | − | + | − | + | + | + | − | + | + | + | − |
| HDV111-1 | O49:H33 | − | − | − | + | − | − | − | − | − | + | − |
| SC289-1 | O49 | − | − | + | + | − | − | + | + | − | + | + |
| SC327-5 | O49 | − | − | + | + | − | − | − | − | − | − | + |
| RN349-5 | O49 | − | + | − | + | − | + | − | − | − | − | − |
| SC389-1 | O49 | − | − | − | − | − | − | − | + | + | + | + |
| MA821-3 | O54 | + | − | − | + | + | − | + | − | − | − | − |
| MA253-1 | O55 | − | − | − | − | − | − | + | − | − | − | − |
| RN513-3a | O66 | − | − | − | + | − | + | − | + | − | + | + |
| MA655-5 | O66 | − | − | − | + | − | − | − | − | − | − | − |
| HSP59-1 | O78:H2 | + | − | − | + | + | − | + | − | − | − | − |
| MA565-1 | O83 | − | − | + | + | − | − | − | + | − | + | − |
| MA697-7a | O86 | − | − | + | + | − | − | − | − | − | − | − |
| RN501-7 | O90 | − | − | + | + | − | − | − | − | + | − | − |
| MA537-1 | O90 | − | − | − | + | − | − | − | + | − | − | − |
| MA703-1 | O90 | + | − | − | + | − | − | + | − | + | − | − |
| HDV117-11 | O92:H2 | − | − | − | + | − | − | + | − | − | − | − |
| HSP131-11 | O117 | − | − | − | + | + | − | + | − | − | + | − |
| RN407-1 | O117 | + | − | − | + | − | − | + | − | + | + | + |
| MA495-2 | O117 | − | − | − | + | − | − | − | + | − | − | − |
| MA567-4 | O125 | + | − | − | + | + | − | − | − | − | − | − |
| MA659-2 | O125:NM | − | − | + | + | − | − | + | + | − | + | − |
| MA823-6 | O125 | + | − | − | + | − | − | − | − | − | − | − |
| RN735-1 | O126:NM | − | − | + | + | − | − | + | + | − | + | − |
| HSP713-1 | O126 | − | − | + | + | − | − | + | + | − | + | − |
| HDV83-1 | O153:H26 | − | − | − | + | − | + | − | + | − | − | − |
| RN747-9 | O155 | + | − | − | + | − | + | + | + | + | + | + |
| MA545-1 | O170 | − | − | − | − | − | − | + | − | − | − | − |
| MA807-5 | O170 | − | − | + | + | − | − | − | − | − | + | − |
| RN733-3 | O174:NM | − | − | + | + | − | + | + | + | + | + | − |
| RN611-3 | ONT | + | − | − | + | − | − | − | + | − | + | + |
| MA693-5 | ONT | − | − | + | + | − | − | − | − | − | + | − |
| MA831-5 | ONT | − | − | + | + | + | − | − | − | − | + | − |
daaC positive.
NM, nonmotile; NT, nontypeable.
TABLE 3.
Distributions of plasmid and chromosomal genes among 32 EAEC probe-positive isolates from controls
| Strain | Serotype or serogroupb | Probe result
|
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Plasmid gene
|
Chromosomal gene
|
|||||||||||
| AAF/I | AAF/II | AAF/III | aggR | aap | astA | pet | shf | shET1/pic | irp2 | hly | ||
| SC272-1 | O15:NM | − | − | + | + | − | − | − | − | − | + | − |
| RN630-1 | O15 | − | − | − | − | − | − | + | + | − | − | − |
| MA524-1 | O17 | − | − | + | + | − | − | − | − | − | + | + |
| RN416-3 | O17:NM | − | + | − | + | − | + | − | + | + | + | − |
| MA496-2 | O36 | − | − | − | + | − | − | − | − | − | − | − |
| RN572-1 | O41 | − | − | + | + | − | − | − | − | − | + | + |
| RN612-1 | O48 | − | − | − | + | − | − | + | − | − | − | − |
| MA670-3 | O48:NM | − | + | − | + | − | + | − | + | − | + | − |
| MA228-4 | O49 | − | − | − | + | − | − | − | + | − | − | − |
| MA232-2 | O49:NM | − | − | + | + | − | + | + | + | − | + | − |
| MA330-4 | O49 | − | − | + | + | − | − | − | − | − | + | − |
| RN620-1 | O49 | − | − | − | + | − | − | − | + | − | + | − |
| RN782-1a | O62 | − | − | − | + | − | − | − | + | − | + | + |
| RN514-3 | O66 | − | − | − | + | − | − | + | + | − | − | − |
| RN776-8 | O73 | − | − | − | + | − | − | + | + | − | − | − |
| MA498-1 | O90 | + | − | − | + | + | − | − | − | − | + | − |
| RN766-2 | O90 | − | − | + | + | − | − | + | − | − | + | − |
| RN220-1 | O92 | − | − | − | + | − | − | − | − | − | − | − |
| MA252-5 | O104 | − | − | + | + | − | − | − | + | − | + | − |
| RN208-9a | O117 | − | − | − | + | − | − | − | + | − | + | − |
| RN190-2 | O125:H21 | − | − | + | + | − | + | + | − | + | + | − |
| RN196-3a | O125:H21 | + | − | − | + | − | + | + | + | − | − | − |
| MA660-5 | O125 | − | + | − | + | − | − | + | − | − | + | − |
| HDV262-8 | O126 | − | − | + | + | − | − | − | + | + | − | − |
| MA566-1 | O150 | − | − | − | + | − | − | − | − | + | − | − |
| RN770-1 | O150:NM | + | − | − | + | − | + | + | + | − | + | − |
| MA478-2 | O153 | − | − | + | + | − | − | + | − | − | + | − |
| HSP166-5 | ONT:H18 | + | − | − | + | − | − | − | − | − | + | − |
| RN460-3 | ONT:NM | + | − | − | + | − | − | − | − | − | + | + |
| MA486-1 | ONT:NM | − | − | + | + | − | + | − | − | + | + | − |
| RN632-3 | ONT | − | − | + | + | − | − | − | + | − | + | − |
| RN784-3 | ONT | + | − | − | + | − | + | − | + | − | − | + |
daaC positive.
NM, nonmotile; NT, nontypeable.
Characteristics of EAEC probe-negative strains.
Twenty-one (75%) EAEC probe-negative strains belonged to 16 distinct serogroups (Table 4). The majority of strains in this group carried two or more of the genes for which assays were conducted, with two being the modal value; but two strains isolated from a control did not test positive for any of the factors. The AAF, aggR, and aap genes were present in only a minority of strains. The astA, pet, shf, ShET1/pic, irp2, and hly genes were found more frequently in the patients than in the controls. The combination astA and shf was found in 16 strains (57%), followed by 7 strains (25%) possessing the combination astA, shf, and irp2.
TABLE 4.
Distributions of plasmid and chromosomal genes among 28 EAEC probe-negative isolates from patients and controls
| Strain source and strain | Serotype or serogroupb | Probe result
|
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Plasmid gene
|
Chromosomal gene
|
|||||||||||
| AAF/I | AAF/II | AAF/III | aggR | aap | astA | pet | shf | shET1/pic | irp2 | hly | ||
| Patients | ||||||||||||
| RN731-5a | O3 | − | − | − | − | − | − | + | − | − | + | + |
| SC385-5 | O42 | − | − | − | − | − | + | − | + | − | − | − |
| HSP53-1 | O42 | − | − | − | + | − | − | − | − | − | − | − |
| SC319-3 | O49:NM | − | − | − | − | − | + | − | + | + | + | − |
| RN779-4 | O54 | − | − | − | − | − | + | − | + | + | − | − |
| SC289-3a | O86:NM | − | − | − | − | − | + | − | + | − | − | − |
| SC323-4 | O89:NM | − | − | − | − | − | + | − | + | − | + | − |
| MA701-1 | O98 | − | − | − | − | − | + | − | + | − | − | − |
| RN453-1 | O117 | − | − | − | + | − | + | + | + | − | + | − |
| RN741-5 | O126:NM | − | − | − | − | − | + | − | + | − | + | − |
| RN153-1 | O126 | − | − | − | − | − | + | − | + | − | − | − |
| RN771-5 | O162:NM | − | − | − | − | − | + | − | + | − | − | − |
| RN749-9 | O162 | − | − | − | − | − | + | + | + | − | + | − |
| HDV137-10 | ONT | − | − | − | − | − | + | + | + | + | + | − |
| RN767-7a | ONT | − | − | − | − | − | − | + | − | + | − | + |
| RN751-9 | ONT | − | − | − | − | − | + | + | + | + | + | − |
| SC779-1 | ONT | − | − | − | − | − | + | − | − | − | − | − |
| SC373-4a | ONT | − | + | − | + | + | + | + | + | − | − | − |
| Controls | ||||||||||||
| SC358-1 | O6:NM | − | − | − | − | − | − | + | − | − | − | + |
| SC378-3a | O28 | − | − | − | + | − | + | − | + | − | − | − |
| SC730-1 | O49 | − | − | − | − | − | − | + | − | − | − | − |
| RN406-5 | O52 | − | − | − | − | − | − | − | − | + | − | − |
| MA558-7 | O66:NM | − | − | − | − | − | − | − | + | − | + | − |
| RN772-3 | O89 | − | − | − | − | − | + | − | + | − | − | − |
| RN502-1a | O141 | − | − | − | − | − | − | − | − | − | − | − |
| RN582-3 | O174:NM | − | − | − | − | − | − | + | − | − | − | − |
| SC796-1 | ONT | − | − | − | − | − | − | − | − | − | + | − |
| SC798-4 | ONT | − | − | − | − | − | − | − | − | − | − | − |
daaC positive.
NM, nonmotile; NT, nontypeable.
Distribution of virulence factors in children with and without diarrhea.
The distribution of EAEC probe-positive and probe-negative strains with the different virulence factors in children with and without diarrhea is presented in Table 5. The common virulence factors found in EAEC probe-positive and probe-negative isolates included the Pet, EAST-1, Irp2, Shf, ShET1/Pic, and Hly virulence markers. AAFs, AggR, and Aap were predominantly identified only in probe-positive strains. The AggR virulence factor was the most common gene marker identified in EAEC probe-positive strains and was found in 74 strains (91%), followed by Irp2, which was found in 49 strains (60%). In this group of strains, the Aap virulence marker was found significantly more frequently (P = 0.023) in isolates from children with diarrhea (22%) than in controls (3%). EAST-1 and Shf were the most frequently detected markers (61%) in EAEC probe-negative strains and were found to be significantly associated with diarrhea (P = 0.003 and P = 0.020, respectively).
TABLE 5.
Distributions of virulence-related markers among EAEC isolates
| Virulence factor | No. (%) of isolates
|
|||||
|---|---|---|---|---|---|---|
| EAEC probe positive
|
EAEC probe negative
|
|||||
| Cases (n = 49) | Controls (n = 32) | Total (n = 81) | Cases (n = 18) | Controls (n = 10) | Total (n = 28) | |
| AAF/I | 9 (18) | 6 (18) | 15 (18) | 0 | 0 | 0 |
| AAF/II | 6 (12) | 3 (9) | 9 (11) | 1 (5) | 0 | 1 (3.6) |
| AAF/III | 18 (37) | 12 (37) | 30 (37) | 0 | 0 | 0 |
| AggR | 43 (88) | 31 (97) | 74 (91) | 3 (17) | 1 (10) | 4 (14) |
| Aap | 11 (22) | 1 (3)a | 12 (15) | 1 (5) | 0 | 1 (3.6) |
| EAST-1 | 13 (26) | 8 (25) | 21 (26) | 15 (83) | 2 (20)b | 17 (61) |
| Pet | 20 (41) | 11 (34) | 31 (38) | 7 (39) | 3 (30) | 10 (36) |
| Shf | 24 (49) | 16 (50) | 40 (49) | 14 (78) | 3 (30)c | 17 (61) |
| ShET1/Pic | 8 (16) | 5 (16) | 13 (16) | 5 (28) | 1 (10) | 6 (21) |
| Trp2 | 28 (57) | 21 (66) | 49 (60) | 8 (44) | 2 (20) | 10 (36) |
| Hly | 9 (18) | 5 (16) | 14 (17) | 2 (11) | 1 (10) | 3 (11) |
Significant at P = 0.023, as determined by χ2 test.
Significant at P = 0.003, as determined by χ2 test.
Significant at P = 0.020, as determined by χ2 test.
DISCUSSION
EAEC has been associated with endemic pediatric diarrhea worldwide. However, the pathogenic mechanisms of EAEC infection are not fully understood. Moreover, there appears to be a significant heterogeneity of virulence markers among EAEC isolates (19). By using EAEC strains isolated in a case-control study, we assessed the prevalence of putative virulence factors, such as AFF/I, AAF/II, AAF/III, AggR, Aap, EAST-1, Pet, Shf, ShET1/Pic, Irp2, and α-Hly, in an attempt to identify their roles as enteric virulence factors.
In our study, a correlation between the presence of a specific EAEC marker and diarrhea was found for both EAEC probe-positive and probe-negative strains. Except for a study conducted in Nigeria (19), in which the presence of genes related to AAF/II was associated with diarrhea in children, none of the pAA markers has been statistically associated with diarrhea, until now. Recently, Piva et al. (20) detected pic sequences significantly more frequently in EAEC probe-positive strains from children with diarrhea than from controls.
Among the EAEC probe-positive strains, we detected the aap gene much more frequently in strains from patients than from controls (P = 0.023). Recently, Sheikh et al. (27) showed that this gene encodes an antiaggregation protein named “dispersin” that is expressed in vivo, highly immunogenic, and present in most EAEC probe-positive strains. Those investigators also proposed that this protein might be representative of a functional class of colonization factors.
Among the EAEC probe-negative strains, astA and shf were found to be significantly associated with diarrhea (P = 0.003 and P = 0.020, respectively). In the present study, 61% of EAEC probe-negative strains from patients harbored the astA gene. A similar astA gene frequency has previously been reported among EAEC probe-negative strains (7), but in that study, EAST-1 was detected more frequently in strains from controls than in strains from patients. Okeke et al. (19) and Rich et al. (22) detected EAST-1 in 23 and 45% of EAEC strains, respectively, but no correlation between the presence of EAST-1 and diarrhea was found. Recently, Piva et al. (20) detected EAST-1 in 73% of EAEC probe-positive strains. The astA gene has been detected not only in EAEC strains but also in EPEC, enterotoxigenic E. coli, Shiga-toxin producing E. coli, and enteroinvasive E. coli strains (6, 28, 32). However, its significance in the pathogenesis of E. coli infection remains unclear.
We also detected shf in 61% of EAEC probe-negative strains. This gene encodes a cryptic open reading frame that shares 25% amino acid identity with the Staphylococcus epidermidis IcaB protein, which is implicated in intercellular adhesion (11). This gene has been found in many EAEC probe-positive strains and a few EAEC probe-negative strains (5, 7).
The majority of the EAEC isolates in this study reacted with one of the antisera used. Other workers have shown that many EAEC strains are O nontypeable (13, 28). The diversity of serotypes seen in this study and the apparent lack of a correlation between a particular serotype and the presence of a particular virulence marker suggest that several clones of EAEC from children with diarrhea may be responsible for diarrhea in different cities in Brazil. It was also interesting that the strains belonging to serogroups O42 and O44 were detected only in children with diarrhea. EAEC isolates of diverse origins have been found to be of these serogroups, and thus, these serogroups could serve as markers for pathogenic EAEC strains.
Few studies have evaluated the prevalence of EAEC markers in EAEC probe-positive and probe-negative strains isolated from subjects in case-control studies. The present study provides evidence that some of the recognized pAA-encoded factors of EAEC (AAF/I, AAF/II, AAF/III, Aap, and AggR) are prevalent in EAEC probe-positive strains and that others (Pet, EAST-1, Irp2, ShET1/Pic and Hly) are found in both groups of EAEC strains. In a recent examination of strains in a case-control study, Elias et al. (7) found that EAEC probe-positive strains isolated in São Paulo exhibited a wide range of virulence characteristics compared to the number exhibited by EAEC probe-negative strains. In that study, they suggested that the EAEC probe-positive strains might have a higher pathogenic potential or, alternatively, that EAEC probe-negative strains may harbor virulence factors that have not yet been described. In contrast, Bouzari et al. (3) found that EAEC probe-negative strains in Iran share virulence factors with EAEC probe-positive isolates. Our findings, along with those of Elias et al. (7), suggest the possibility that EAEC probe-positive strains comprise a distinct subcategory of EAEC that could be called typical EAEC.
Recently, Jiang et al. (14) suggested that the aggA and aggR genes alone or in combination with other virulence factors (aafA or aap) might be used to identify pathogenic EAEC strains. In the present study, both aggR and aggA were found at similar frequencies in patients and controls.
In conclusion, our data reinforce the heterogeneity of EAEC strains with regard to both pAA plasmid-borne and chromosomal factors. However, we have apparently identified two virulent subpopulations of EAEC: EAEC probe-positive strains carrying the aap gene and EAEC probe-negative strains harboring the astA and the shf genes. Furthermore, our data suggest that aggR can be used as an important genetic marker for EAEC probe-positive strains.
Acknowledgments
We thank Chantal Le Bouguénec for the gift of the AAF/III reference strain.
This work was funded by the Conselho Nacional de Desenvolvimento Científico e Tecnológico and the Fundação de Amparo à Pesquisa do Estado de São Paulo.
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