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Enteroaggregative Escherichia coli Virulence Markers: Positive Association with Distinct Clinical Characteristics and Segregation into 3 Enteropathogenic E. coli Serogroups

  1. Alex L. Pereira1,
  2. Lúcia R. Ferraz2,
  3. Rejane S. N. Silva2 and
  4. Loreny G. Giugliano1
  1. 1Laboratório de Microbiologia, Departamento de Biologia Celular, Universidade de Brasília, Brasilia, Brazil
  2. 2Laboratório Central de Saúde Pública do Distrito Federal, Brasilia, Brazil
  1. Reprints or correspondence: Dr. Loreny G. Giugliano, Laboratório de Microbio-logia, Departamento de Biologia Celular, Instituto de Biologia, Universidade de Brasília, 70910-900, Brasília-DF (giuglian{at}unb.br).
 
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Abstract

Background. Enteroaggregative Escherichia coli (EAEC) are a heterogeneous category of emerging pathogens associated with acute and persistent diarrhea.

Methods. We performed a case-control study to evaluate the association of EAEC strains and their virulence markers with infantile diarrhea in Brasília, Brazil.

Results. Of the EAEC markers that were tested, only EAEC heat-stable toxin (EAST1), protein involved in colonization (Pic), and the CVD432 sequence showed positive correlation with diarrhea; however, these virulence traits presented associations with distinct clinical characteristics. Although CVD432-positive strains were associated with persistent diarrhea, EAST1-positive strains were statistically correlated with acute diarrhea only in children >6 months of age. Typical EAEC strains were found with equal frequency in case patients and control subjects. O antigens of enteropathogenic E. coli (EPEC) were also sought, and the data showed that the EAEC markers are frequently segregated into 3 EPEC serogroups: O126, O128, and O158.

Conclusions. The presence of EPEC O antigens along with EAEC markers influenced the positive association of E. coli strains with diarrhea. Concerning the detection of the AA phenotype, the CVD432 sequence presented a low positive predictive value, regardless of its high specificity as a marker.

Enteroaggregative Escherichia coli (EAEC) is defined by its ability to adhere to cultured cells in a “stacked brick” appearance [1]. This phenotype, termed “aggregative adherence” (AA), is associated with specific fimbriae (AAFs) encoded by plasmids (pAAs) [2, 3]. Two major fimbriae, known as “AAF/I” and “AAF/II,” have been described in prototype EAEC strains 17-2 and 042, respectively. Other factors are associated with this adherence pattern, indicating its multifactorial nature [4, 5]. Several toxins expressed by EAEC strains have already been identified. Among the well-known toxins are EAEC heat-stable toxin (EAST1) and plasmid-encoded toxin (Pet), both of which are encoded by pAAs [6–8]. Furthermore, a serine protease involved in the colonization process (Pic) is encoded by the chromosome from strain 042 [9].

A cryptic sequence known as “CVD432,” which is isolated from the pAA, has been used as an EAEC molecular marker in epidemiological studies since its discovery [10]. Nowadays, it is known that the CVD432 sequence comprises the locus att that encodes an ABC transporter system. A transcription activator known as “AggR,” the gene of which lies on pAAs, has been described as the major EAEC virulence regulator. AggR controls diverse virulence genes encoded by pAAs as well as by chromosomes, showing the presence of a pAA-associated regulon in EAEC strains.

Historically, enteropathogenic E. coli (EPEC) strains were associated with specific serogroups that were involved in outbreaks of infantile diarrhea during the 1950s [11, 12]. However, many current studies have shown that EAEC strains share classical O antigens of EPEC [13, 14]. This fact has encouraged scientists to attempt to detect EPEC strains on the basis of findings of phenotypic and molecular assays [15]. The serogroups O111, O125, O126, and O128 are among the traditional EPEC serogroups frequently shared by EAEC strains [14, 16].

EAEC has been recognized as an emerging pathogen in both developed and developing countries [17]. Epidemiological studies performed in economically underprivileged communities showed that EAEC strains were strongly associated with persistent diarrhea in children [18–20]. Other studies also revealed the involvement of EAEC with outbreaks of acute diarrhea affecting newborns and children in industrialized countries [21–23]. Recently, some epidemiological studies have suggested that CVD432-positive strains, which are predicted to carry the AggR regulon, are the true EAEC pathogens. Therefore, the term “typical EAEC” has emerged to denote E. coli strains that harbor pAAs [24–26]. Despite the best efforts, controversial data generated in different geographic areas have made the epidemiological profile of EAEC quite difficult to understand.

We conducted a case-control study involving children, to determine the role of EAEC strains and their virulence traits in infantile diarrhea. Our results allow us to conclude that specific EAEC markers are associated with distinct clinical characteristics. Furthermore, among E. coli strains positive for classical O antigens of EPEC, EAEC virulence traits are primarily segregated into 3 major serogroups.

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Materials and Methods

Specimens and strains. E. coli strains were isolated from newborns and from children up to 5 years of age who were being treated at 2 hospitals in Brasília, Brazil (Hospital Universitário de Brasília and Hospital Materno-Infantil de Brasília). One hundred thirty-four fecal specimens were collected from patients with diarrhea, and 127 fecal specimens were collected from healthy children. Control subjects were defined as children who did not present with diarrhea in the 4 weeks before sample collection. The subjects were matched by age and socioeconomic class, within 15 days of sample collection. Subjects who experienced any carbohydrate intolerance or congenital malformation of the gastrointestinal tract were excluded from the study, as were subjects who had used antibiotics within 15 days before sample collection. Diarrhea was characterized by an increased number of evacuations with loose feces. “Persistent diarrhea” was defined as diarrhea lasting ≥14 days, in accordance with World Health Organization guidelines. In general, 5 E. coli strains were isolated from each fecal sample, with a total of 1253 isolates recovered. Salmonella and Shigella organisms were sought in all samples and were isolated concomitantly with E. coli strains. All E. coli strains underwent serological assays for the detection of classical serogroups of EPEC. Bacterial strains were preserved at −20°C in Luria-Bertani broth medium with 15% glycerol.

HeLa cells and adhesion assays. All bacteria used in ad-hesion assays were cultured in Luria-Bertani broth medium at 37°C for 18 h. HeLa cells were cultured in Dulbecco's modified Eagle medium (DMEM; Gibco BRL) with 10% fetal bovine serum and antibiotics in an atmosphere with 4% CO2 at 37°C. Adhesion assays were performed as described by Mathewson et al. [27], with modifications. HeLa cells (0.6×105 cells/mL) were cultured on glass coverslips by use of 24-well plate culture (600 µL/well) (Costar). Cells were grown to 50%–70% confluence, and the medium then was changed to DMEM supplemented with 1.4% mannose and 2% fetal bovine serum (600 µL/well). Then, 200 µL of bacterial culture was used to infect the cells for 6 h. After infection, the wells were washed 5 times with Dulbecco's PBS, and the cells were fixed with methanol, stained with May-Grünwald and Giemsa stains, and analyzed using light microscopy. EAEC strain 042 was used as the positive control for the aggregative phenotype.

Detection of virulence markers. Virulence markers were detected by polymerase chain reaction performed using the primers listed in table 1 [28–32]. Multiplex reactions were developed to detect markers for EAEC (CVD432, AAF/I, Pet, and Pic) and EPEC. With regard to EAEC markers, the cycling conditions for multiplex polymerase chain reaction were as follows: 10 cycles at 94°C for 1 min, at 55°C for 1 min, and at 72°C for 90 s, followed by 20 cycles at 94°C for 1 min, at 60°C for 1 min, and at 72°C for 90 s. EPEC markers, AAF/II, and EAST1 were amplified using similar parameters, except for the melting temperature (52°C). Supernatants derived from bacterial suspension treated by boiling were used as the source of DNA template.

Table 1
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Table 1

Primers used in polymerase chain reaction analysis.

Statistical analysis. Results were analyzed using the χ2 test or Fisher's exact test, in accordance with the limitations of both tests in relation to the analyzed sample. Results for which P≤.05 were considered to be statistically significant.

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Results

Patients and characterization of diarrhea. To investigate the role of EAEC virulence markers in diarrhea, we performed a case-control study involving 134 fecal samples collected from case patients and 127 fecal samples collected from control sub-jects. Only 12 (8.9%) of the samples obtained from patients were found to be positive for Salmonella or Shigella strains, and 24 patients (17.9%) were considered to have persistent diarrhea. Two cases of persistent diarrhea (8.3%) were found to be associated with Salmonella or Shigella infection; both cases developed in children <6 months of age. Analyses focusing on the association of EAEC markers with diarrhea were conduct-ed, excluding Salmonella- or Shigella-positive samples. E. coli strains harboring EAEC markers were frequently found to colonize the children in all groups analyzed (36.9% of the samples). Forty-five percent of the fecal samples obtained from patients with acute diarrhea were found to have E. coli strains positive for EAEC markers (P<.05, compared with control subjects), whereas among patients with persistent diarrhea and control subjects, the frequency of such strains was 27.2% and 32.2%, respectively (table 2). Analyses performed to detect specific virulence traits showed that EAST1-positive (EAST1+) samples accounted for 27% of the samples collected from patients with acute diarrhea. This frequency was statistically significant, compared with that noted for the control group (P<.05). On the other hand, the frequencies of EAST1+ samples collected from patients with persistent diarrhea and control subjects were not statistically different.

Table 2
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Table 2

Virulence markers in the patients and control subjects studied.

Virulence traits associated with acute and persistent diarrhea. To determine associations of EAEC markers with diarrhea, 1194 E. coli strains were tested, and the frequencies of virulence markers were determined (table 3). With regard to EAEC markers, several combinations (genotypes) were found in the strains studied. Nevertheless, some of these genotypes were rarely found and did not present any relevance for the epidemiological study; therefore, they are not shown in the results. With the exception of EAST1, the remaining factors were detected with equal frequency in patients and control subjects (table 3). Detection of the astA gene occurred statistically more frequently in patients than in control subjects; this virulence marker was detected in 16.7% of strains isolated from patients and in only 7.9% of strains isolated from control subjects (P<.001). Likewise, this tendency held in analyses considering genotype traits. EAST1+ strains and CVD432+EAST1+ strains (which tested positive for both markers) showed a significant association with diarrhea (P<.001 and P<.05, respectively) (table 3).

Table 3
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Table 3

Distribution of enteroaggregative Escherichia coli (EAEC) markers in E. coli strains isolated from children.

Similar analyses were performed that highlighted 89 E. coli strains recovered from patients with protracted diarrhea and 485 strains recovered from patients with acute diarrhea. This approach showed that the CVD432 sequence was associated with persistent diarrhea, because its frequency was significantly higher in this clinical group than in patients with acute diarrhea (P<.001) or control subjects (P<.01) (table 3). On the other hand, the astA gene was strongly associated only with acute diarrhea (P<.001). Likewise, the CVD432+ genotype was associated with persistent diarrhea, whereas the EAST1+ genotype was associated only with acute diarrhea. It is interesting to note that 5.6% of strains recovered from patients with persistent diarrhea harbored the CVD432 and EAST1 markers exclusively, whereas this genotype trait was observed in only 0.8% and 0.3% of strains recovered from patients with acute diarrhea and control subjects, respectively (P<.01). Moreover, no E. coli strain isolated from patients with persistent diarrhea showed a positive result for the tested fimbriae, in spite of the high frequency of occurrence of CVD432 in strains isolated from these patients.

Influence of age on the association of EAEC markers with diarrhea. To clarify whether associations of EAEC markers with diarrhea are influenced by age, analyses were performed in which patients and control subjects were stratified into 2 age groups: children <12 months of age and children >12 months of age (table 4). Although the frequency of CVD432 was similar among the strains isolated from patients and control subjects in both age groups studied, E. coli strains with the CVD432+ genotype showed a positive association with diarrhea in children <12 months of age (P<.05). The frequency of the EAST1 marker, as well as the presence of EAST1+ strains, was statistically associated with diarrhea in both age groups. Despite the steady association of EAST1+ strains with diarrhea shown so far, analyses performed with a more narrow patient age range showed that EAST1+ E. coli strains had a positive association with diarrhea only in children >6 months of age (P<.01). In children 6–12 months of age, the frequency of EAST1+ strains was 13% (28 of 214 samples) and 4% (9 of 220 samples) in patients and control subjects, respectively (P<.001). Nevertheless, among children <6 months of age, EAST1+ strains were isolated from patients (9 [8.2% ] of 109 samples) and control subjects (5 [4.2%] of 118 samples) with similar frequency (P=.272).

Table 4
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Table 4

Distribution of enteroaggregative Escherichia coli (EAEC) markers in E. coli strains isolated from 2 distinct groups of children with acute and persistent diarrhea.

Likewise, the frequency of EAEC markers was analyzed in patients with acute and persistent diarrhea (table 4). When patients with persistent diarrhea were compared with control subjects, the frequency of CVD432-homologous sequence was statistically significant only in isolates recovered from children with persistent diarrhea who were <12 months of age. This difference was due to the high frequency of the CVD432+ genotype among the strains isolated from patients with protracted diarrhea, compared with strains isolated from control subjects (P<.001) and patients with acute diarrhea (P=.05). With regard to children >12 months of age, it is interesting to note that although the frequency of the CVD432 sequence was statistically equal in control subjects and patients with persistent diarrhea, E. coli strains with the CVD432+EAST1+ genotype accounted for 11.6% and 0.7% of strains recovered from patients with persistent diarrhea and control subjects, respectively (P<.001). This difference held when we conducted the same analysis comparing patients with persistent diarrhea with those with acute diarrhea (P<.001). The EAST1 marker was associated only with acute diarrhea in both age groups analyzed (P<.001). Finally, E. coli strains that tested positive only for Pic were associated with acute diarrhea in children <12 months of age (P=.05).

The AA pattern and its correlation with the CVD432 sequence. A total of 191 E. coli strains, including 24 strains isolated from patients with Salmonella- or Shigella-associated diarrhea, were tested in 6-h adhesion assays. The adhesion assays showed that diffusely adherent E. coli (DAEC) was the most frequently noted pathotype (47.6%), followed by EAEC (25.6%). Of 49 EAEC strains detected, 37 (75.5%) were positive for at least 1 of the EAEC markers tested. It is interesting to report that only 20 (40.8%) of 49 EAEC strains showed a CVD432-homologous sequence and, therefore, were considered to be typical EAEC. Otherwise, of the 142 strains that did not display the AA phenotype, 12 strains presented the CVD432 sequence (specificity, 91.5%). Concerning detection of the AA pattern, the positive and negative predictive values of the CVD432 marker were 65.7% and 83.3%, respectively.

EAEC strains accounted for 11 (33.3%) of 33 strains and 11 (17.1%) of 64 strains isolated from patients with persistent and acute diarrhea (P=.071), respectively; for control subjects, this frequency was 24.2% (17 of 70 strains). With regard to typical EAEC, strains in this category were found with similar frequency in patients and control subjects. Typical EAEC ac-counted for 10 (45.4%) of 22 EAEC strains isolated from pa-tients and for 10 (58.8%) of 17 EAEC strains isolated from control subjects. The frequency of typical EAEC in patients with persistent and acute diarrhea was 27.2% (3 of 11 strains) and 63.6% (7 of 11 strains), respectively.

O antigens of EPEC, EAEC markers, and the AA pattern. Serological assays were conducted with 1257 E. coli strains, to estimate the frequency of O antigens of EPEC. Of the 223 strains positive for EPEC serogroups (OEPEC+), 38 strains, which belonged to serogroups O86, O114, O119, O126, O128, and O158, hosted ≥1 type of the EAEC markers analyzed. On the other hand, isolates belonging to the serogroups O26, O55, O111, O125, O127, and O142 (n=83) did not show positive results for the tested EAEC markers. Of the OEPEC+ strains that hosted EAEC markers, 30 (78.9%) of 38 strains belonged to serogroups O126, O128, or O158 (table 5). All 68 strains presenting antigens O126, O128, or O158 were also tested for the presence of EPEC markers eae and EAF; however, none of the strains showed any positive result.

Table 5
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Table 5

Enteropathogenic Escherichia coli serogroups positive for enteroaggregative E. coli (EAEC) markers.

To estimate the frequency of EAEC among the strains positive for EPEC serogroups, 61 OEPEC+ strains, mainly one isolate from each sample, were tested in adhesion assays. The tests showed that 22.9% of the OEPEC+ strains presented aggregative adhesion. These EAEC strains were positive for the following antigens: O55 (n = 1), O111 (n = 1), O125 (n = 1), O126 (n = 3), O128 (n = 2), O142 (n = 1), and O158 (n = 5). Furthermore, strains positive for O126, O128, and O158 accounted for 10 (71.4%) of 14 of the EAEC strains belonging to EPEC serogroups. Similar analysis showed that, among DAEC, these sero-groups were found in only 17.2% of the strains. Of all the strains tested in adherence assays, only 2 strains that belonged to serogroup O111 presented localized adhesion.

Influence of O antigens on the association of EAEC markers with diarrhea. Despite the fact that OEPEC+ strains were found with similar frequencies in patients and control subjects, OEPEC+ strains that hosted CVD432 or EAST1 sequences were statistically associated with diarrhea (table 6). It is important to note that the previous case-control analysis that was based exclusively on CVD432 sequence did not indicate any positive correlation of this marker with diarrhea.

Table 6
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Table 6

Frequency of enteropathogenic Escherichia coli serogroup O and enteroaggregative E. coli (EAEC) virulence markers in strains isolated from children.

Analyses focusing on specific serogroups revealed that O128-positive strains showed statistical correlation with diarrhea (P<.01). In addition to this fact, 61.1% of these strains isolated from patients presented EAEC markers; however, no strains isolated from control subjects presented such features.

Although serogroup O158 had been noted to have similar frequencies in patients and control subjects, O158 E. coli strains hosting EAEC markers accounted for 53.3% of the strains isolated from patients, whereas the frequency of such strains in the control subjects was 6.6% (P<.01). E. coli strains that belonged to serogroup O126 and were positive for EAEC markers were found equally in patients and control subjects.

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Discussion

EAEC was described in the late 1980s, and, since that time, many studies have been conducted to illuminate its epidemiological profile and pathogenesis. In our study, children were frequently colonized by EAEC+ strains. Fecal samples that were positive for EAEC+ strains were statistically associated with acute diarrhea but not with persistent diarrhea. When specific markers were highlighted, samples positive for strains harboring the EAST1 gene were associated only with acute cases. Despite CVD432-positive fecal samples not having been statistically associated with any patient groups, the highest frequency of CVD432-positive strains (22.7%) was noted in samples collected from patients with persistent diarrhea. Nowadays, it is known that the EAST1 gene is frequently found in other diarrheogenic categories of E. coli, such as EPEC, DAEC, and enterohemorrhagic and enterotoxigenic E. coli. In fact, the high frequency of astA among E. coli pathotypes is explained by the constant association of this gene with insertion sequence-homologous elements [33].

Case-control analysis based on genotype characterization of isolated strains endorsed the correlation of the EAST1 gene with acute diarrhea and showed that distinct genotype pat-terns can be associated with different clinical characteristics. Although EAST1+ strains were strongly associated with acute diarrhea, CVD432+ and CVD432+EAST1+ strains were associ-ated solely with persistent diarrhea. Although its biological function has remained unknown for long time, the CVD432 sequence has been used as an EAEC marker since its discovery. Nishi et al. [34] showed that the CVD432 sequence constitutes the aat locus that is responsible for the expression of an ABC transporter in EAEC strain 042. Coupled with this fact, 86% of tested, att-positive strains also hosted the aggR gene. Thus, in contrast with EAST1+ strains, CVD432-harboring strains, if not classified as EAEC, present a genetic profile closely associated with this E. coli category.

The age-stratified analyses showed that different CVD432-related genotypes were associated with persistent diarrhea in the 2 groups studied. CVD432+ strains were associated with persistent diarrhea in children <12 months of age, whereas, in children >12 months of age, the genotype associated with pro-tracted diarrhea was CVD432+EAST1+. Regarding EAST1+ strains, this genotype was statistically associated only with acute diarrhea in both age groups analyzed. However, despite the steady association of EAST1 with acute diarrhea, an analysis performed using a more narrow range of age showed the loss of positive correlation of this virulence marker with diarrhea in children <6 months of age. The loss of the positive correlation of EAST1+ strains with diarrhea may be associated, in part, with the immature stages of intestinal development, in which the response to EAST1 toxin is down-regulated. A study performed with an EAST1-sensible mouse lineage demonstrated that 4-day-old suckling mice did not have diarrhea when challenged with an EAST1 preparation [35]. Similar findings have been demonstrated with other enterotoxins [36, 37]. In this study, Pic was associated with acute diarrhea, endorsing the findings of our previous study that showed that Pic-positive strains are potentially involved with infantile diarrhea [28].

In an attempt to illuminate the epidemiological profile of EAEC, and taking into consideration the recognized importance of AggR regulon, the term “typical EAEC” has emerged to indicate the true pathogens among EAEC strains. In this study, typical EAEC strains were found with similar frequency in patients and control subjects. However, when the patients were divided according to type of diarrhea (acute or persistent), the frequency of typical EAEC was higher in patients with acute diarrhea (63.6%) than in patients with persistent diarrhea (27.2%).

Nowadays, it is well known that EAEC strains share classical EPEC serogroups. Recently, Campos et al. [15] revised the frequency of diarrheogenic E. coli categories among 805 strains belonging to traditional EPEC serogroups. In this study, EAEC was the second most frequently detected diarrheogenic category; only typical EPEC was more frequently detected [15]. In fact, serogroups reflect genomic traits that grouped closely related strains; however, O antigens themselves have little or perhaps no influence on the virulence of these strains. The analysis of 223 OEPEC+ strains showed that EAEC virulence markers are frequently segregated into serogroups O126, O128, and O158. These 3 serogroups accounted for 78% of the OEPEC+ strains that tested positive for EAEC markers and for 71% of the OEPEC+-belonging EAEC strains. In addition, among strains O126, O128, and O158, the frequency of the CVD432 marker was 32.3% (22 of 68 strains), whereas among strains O86, O114, and O119, the frequency of the same marker was 3.7% (3 of 79 strains) (P<.001). Why strains grouped into specific serogroups are inclined to harbor specific virulence genes is a question that needs to be clarified. On the other hand, the influence of the association between the EPEC serogroup and the EAEC marker on the involvement of E. coli strains with diarrhea was evaluated in our work. Despite case-control analysis based only on the CVD432 sequence not showing any association of this marker with the disease, OEPEC+ strains positive for CVD432 were statistically associated with the cases. Furthermore, EAEC marker-positive O158 strains were shown to be associated with diarrhea (P<.01), regardless of the similar frequency of this antigen in patients and control subjects. Some studies have already shown that EAEC strains can belong to the serogroups O126 and O128 [13, 14, 38], among other serogroups; however, it seems that this is the first report pointing out serogroup O158 as a potential EAEC serogroup involved with infantile diarrhea. In contrast with our findings, a recent study suggested O158 as a nonpathogenic serogroup [39]. These divergences show that a specific serogroup may cluster genetically heterogeneous strains. Because of controversial data, in some countries, serogrouping has been considered to be an outmoded method for identification of diarrheogenic strains [39, 40].

In conclusion, EAEC strains can present a multitude of virulence gene combinations and, therefore, must be considered a heterogeneous category of potential pathogens. In addition, the occurrence of EPEC O serogroups can influence the association of EAEC marker-positive strains with diarrhea.

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Footnotes

  • Potential conflicts of interest: none reported.

  • Financial support: Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq (grants 520674/99-5).

  • Received July 6, 2006.
  • Accepted September 20, 2006.
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  1. J Infect Dis. (2007) 195 (3): 366-374. doi: 10.1086/510538
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