Abstract: Escherichia coli strains that cause nonbloody diarrhea in infants are known to present four distinct patterns of adherence to epithelial cells, namely, Localized (LA), Localized Adherence Like (LAL), Diffuse (DA) and Aggregative (AA) adherence. Strains with LA and AA are well recognized as a cause of diarrhea, but the role of strains with DA is controversial and strains with LAL have been more frequently isolated in diarrheal than asymptomatic cases. To determine the distribution of the different types of enteropathogenic E. coli (EPEC) adherence patterns in diarrhea, we studied 191 EPEC strains isolated from infants less than 5 years of age with and without diarrhea in Iran. Totally 131 (68.5%) strains adhered to HeLa cells and 60 (31.5%) isolates did not. The results revealed that Localized Adherence (LA) was manifested by 30 of 111 (86%) strains isolated from diarrheal cases of which the most belonged to serogroups O86 and O55. Localized Adherence like (LAL) was exhibited by 16 and Aggregative Adherence (AA) by 9 strains isolated from patients. However, Diffuse Adherence (DA) was exhibited by 8 strains equally distributed between both diarrheal and healthy persons. Undefined Pattern (UDP) was observed in 24 strains. Overall, the results showed that LA, LAL and AA adherence significantly associated with diarrhea (p<0.05).
INTRODUCTION
Enteropathogenic Escherichia coli (EPEC) is a leading cause of infantile diarrhea in developing countries. In industrialized countries, the frequency of these organisms has decreased, but they continue to be an important cause of diarrhea (Nataro and Kaper, 1998, Trabulsi et al., 2002). The central mechanism of EPEC pathogenesis is a lesion called attaching and effacing (A/E), which is characterized by microvilli destruction, intimate adherence of bacteria to the intestinal epithelium, pedestal formation and aggregation of polarized actin and other elements of the cytoskeleton at sites of bacterial attachment. Escherichia coli strains that cause nonbloody diarrhea in infants are known to present three distinct patterns of adherence to epithelial cells, namely, (i) Localized Adherence (LA), where micro colonies attach to one or two small areas on the cells (HeLa or HEp-2); (ii) Diffuse Adherence (DA), where bacteria cover the cells uniformly (Scaletsky et al.,1984, 2005) and (iii) enteroadherent-aggregative adherence (AA), where the bacteria have a characteristic stacked-brick-like arrangement on the surface of the cells and on the glass slide free from the cells (Nataro et al., 1987). The LA pattern is significantly associated with diarrhea production (Nunes et al., 2003) and has been shown to be an important property of enteropathogenic E. coli (EPEC). The LA phenotype is associated with the induction of the attaching and effacing lesions (A/E) (Moon et al., 1983). The pathogenic role of E. coli showing a DA pattern (DAEC) in the etiology of diarrheal disease is controversial (Arikawa et al., 2005; Gunzburg et al., 1993; Jallat et al., 1993). The role of E. coli giving an aggregative pattern of attachment to tissue culture cells (EAEC) in diarrhea has been more frequently related to persistent and pediatric diarrhea (Harrington et al., 2006; Cravioto et al., 1991). EAEC strains possess a plasmid of 60 MDa which is necessary for the expression of the aggregative phenotype (Moon et al., 2005). A new adherence pattern called the Localized Adherence-like (LAL) pattern has described by Scaletsky et al. (1996, 1999). This pattern is characterized by the presence of less-compact microcolonies or clusters of bacteria in a few cells observed only in tests with prolonged incubation periods (6 h). The mechanism of this adherence pattern is unknown. The role of E. coli showing an LAL pattern as an agent of diarrhea is not established yet (Nataro and Kaper, 1998). Studies evaluating the epidemiological importance of HeLa cell adherence pattern of EPEC in diarrheal diseases are required particularly in developing countries. For this reason, we determine the prevalence of the different types of EPEC adherence patterns in strains isolated from diarrheal and asymptomatic children.
MATERIALS AND METHODS
Bacterial strains: A total of 191 EPEC strains were studied for determine the distribution of the different types of adherence patterns. The 111 EPEC strains were isolated from children with diarrhea and 80 strains from children without any gastrointestinal symptoms.
EPEC serogrouping: E. coli strains were isolated on MacConkey plates. Four separate lactose-fermenting and two non-lactose-fermenting colonies of each distinct morphologic type, presumed to be E. coli by colony morphology, were cultivated for biochemical confirmation of species. E. coli colonies were submitted to slide agglutination with polyvalent and monovalent antisera (Bio-Rad Co), against O antigens of EPEC serogroups. The serogroups considered were as follows: O26, O55, O86, O111, O114, O119, O124, O125, O126, O127, O128 and O142. When two or more strains of identical serogroup were isolated from the same infant, only one strain have used in this study.
HeLa cell adherence assay: EPEC colonies were characterized by the pattern of adherence to HeLa cells (National cell Bank of Iran, Institute Pasteur of Iran) in the presence of D-mannose as assayed by the method described by Scaletsky et al. (1984). Three to five individual isolates from each case were assessed for adherence to HeLa cells. Briefly, monolayer HeLa cells infected with bacteria were grown to 50% on coverslips in Leighton tubes in presence of 1% mannose for 3 h at 37°C 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 LA, LAL, DA, or AA patterns under a light microscope. A number of strains adhered in non-specific manner and were put in a different category as Undefined Pattern (UDP). When the adherence pattern was weak or negative, a new preparation was made and examined after a 6 h incubation period. Prototype EPEC strain E2348/69 (serotype O127: H6), E. coli strain E17-2 (serotype O3:H2) and E-coli strain C1845 (serotype O75: NM) showing LA, AA and DA, respectively, was used as positive controls in adherence to HeLa cells assays and E. coli K12 as a negative control.
Statistical analysis: The χ2 test and Fisher’s exact test were used for statistical analysis.
RESULTS
The prevalence of enteropathogenic E. coli serogroups identified in the stool cultures of patients and controls is presented in Table 1. Only the EPEC strains isolated from the stools as a single potential enteropathogenic agent were studied. EPEC serogroups was isolated as sole pathogen from 58.1% (111 of 191) of children with diarrhea, compared with 41.9% (80 of 191) of those without diarrhea (p<0.05). EPEC strains belonged to O26, O55, O86, O111, O114, O119, O125, O126, O127, O128 and O142 serogroups (Table 1).
Totally 131(68.5%) strains adhered to HeLa cells and 60 (31.5%) isolates did not. Four distinct patterns of adherence were distinguished: LA, DA, AA and the LAL pattern, which was observed only in strains incubated for 6 h (Fig. 1). LA was the most frequent pattern among isolates (18.3%), followed by LAL (14.1%), AA (7.9%) and DA (4.2%) (Table 2).
| Table 1: | Prevalence of EPEC serogroups in infants with diarrhea and
healthy cases |
| Table 2: | Adhesion pattern of EPEC strains isolated from patients and
healthy cases |
| UDP = Undefined Pattern, NA = Non Adherence | |
| Fig. 1: | HeLa cell adherence patterns. A: Localized adherence (LA),
B: Localized adherence-Like (LAL), C: Diffuse adherence (DA), D: Aggregative
adherence (AA), E: Undefined adherence (UDP), F: Non-adherence (NA) |
Strains with LA were significantly associated with diarrhea (30 of 35[86%] versus 5 of 35 [14%] in healthy persons; p<0.05). Out of 111 strains isolated from patients, 57 strains (51.3%) were adhered to HeLa cell with a defined pattern (LA, LAL, DA, AA) and 33 (29.7%) showed an undefined pattern (UDP) and the remaining 21 (18.9%) were non-adherent (NA) (Table 2). Among adherence patterns, LA, LAL and AA found to be significantly associated with diarrhea (p<0.05).
Out of the 80 strains isolates from healthy persons, 28 (35%) strains were adherent with a defined pattern and 39 (48.8%) strains were non-adherent. UDP was shown by 13 strains (Table 2).
DISCUSSION
Adherence to HeLa and HEP-2 tissue culture cells have been used as a marker of virulence of EPEC strains (Nakazato et al., 2004; Forestier et al., 1996; Gonzalez et al., 1997). The present results support the evidence from prospective case control studies showing an association between strains with the LA pattern and diarrhea (Cravioto et al., 1991; Gonzalez et al., 1997).
| Table 3: | Distribution of different patterns of adherence among EPEC
serogroups |
Present study and several case-control investigations have showed that LA-positive EPEC were more often isolated from diarrheal cases (Nataro et al.,1985; Baudry et al., 1990). The LA adherence pattern was associated with some EPEC serogroups (Scaletsky et al., 1999; Gomes et al.,1989). In present study, the most common serogroups with LA patterns were O86 and O55 (Table 3), which also was found in other studies (Cravioto et al., 1991; Levine et al., 1988). There was a significant association between strains with LAL pattern and diarrhea in our study (p<0.05). The LAL pattern was found in strains belonging to O114, O111 and O127 classical serogroups (Table 3).
Several studies have implicated DAEC strains as agents of diarrhea (Germani et al., 1996; Scaletsky et al., 2002), while other studies have not recovered DAEC strains more frequently from diarrheal patients than asymptomatic controls (Gunzburg et al., 1993; Germani et al.,1996). In this study, there was no correlation between DA pattern and diarrhea.
E. coli strains that exhibited AA pattern were strongly associated with enteric diseases in Iran and other developing countries (Bouzari et al., 1994; Aslani et al., 1999; Beutin et al., 2003). In our study, E. coli showing an AA pattern was found more frequently in patients (60%) than healthy cases (40%) (p<0.05). The AA isolates belonged to the different EPEC serogroups (Table 3), a finding that has been demonstrated by other authors (Nataro and Kaper, 1998; Scaletsky et al., 2002).
In conclusion, this study showed the importance of adherence virulence factor in EPEC with LA, LAL and AA patterns. There was a significant correlation between these patterns and diarrhea (p<0.05). The existence of the LAL adherence pattern observed in patients with diarrhea may indicate a new mechanism of production of the disease. Further studies are necessary to confirm the present observation as well as to elucidate the pathogenesis of this new type of enteric infection. Present study indicated that EPEC strains with adherence property are more related to diarrhea than the non-adherence ones.
ACKNOWLEDGMENTS
We thanks Dr. H. Shojaei and Dr. S. Bouzari for useful discussion. Mr. H. Khamse and Mr. N. Islami are acknowledged for their technical help.