First isolation and further characterization of enteropathogenic Escherichia coli (EPEC) O157:H45 strains from cattle

Background Enteropathogenic Escherichia coli (EPEC), mainly causing infantile diarrhoea, represents one of at least six different categories of diarrheagenic E. coli with corresponding distinct pathogenic schemes. The mechanism of EPEC pathogenesis is based on the ability to introduce the attaching-and-effacing (A/E) lesions and intimate adherence of bacteria to the intestinal epithelium. The role and the epidemiology of non-traditional enteropathogenic E. coli serogroup strains are not well established. E. coli O157:H45 EPEC strains, however, are described in association with enterocolitis and sporadic diarrhea in human. Moreover, a large outbreak associated with E. coli O157:H45 EPEC was reported in Japan in 1998. During a previous study on the prevalence of E. coli O157 in healthy cattle in Switzerland, E. coli O157:H45 strains originating from 6 fattening cattle and 5 cows were isolated. In this study, phenotypic and genotypic characteristics of these strains are described. Various virulence factors (stx, eae, ehxA, astA, EAF plasmid, bfp) of different categories of pathogenic E. coli were screened by different PCR systems. Moreover, the capability of the strains to adhere to cells was tested on tissue culture cells. Results All 11 sorbitol-positive E. coli O157:H45 strains tested negative for the Shiga toxin genes (stx), but were positive for eae and were therefore considered as EPEC. All strains harbored eae subtype α1. The gene encoding the heat-stable enterotoxin 1 (EAST1) was found in 10 of the 11 strains. None of the strains, however, carried ehx A genes. The capability of the strains to adhere to cells was shown by 10 strains harbouring bfp gene by localized adherence pattern on HEp-2 and Caco-2 cells. Conclusion This study reports the first isolation of typical O157:H45 EPEC strains from cattle. Furthermore, our findings emphasize the fact that E. coli with the O157 antigen are not always STEC but may belong to other pathotypes. Cattle seem also to be a reservoir of O157:H45 EPEC strains, which are described in association with human diseases. Therefore, these strains appear to play a role as food borne pathogens and have to be considered and evaluated in view of food safety aspects.

The main mechanism of EPEC pathogenesis is the ability to introduce attaching-and-effacing (A/E) lesions on intestinal cells characterized by microvillus destruction, intimate adherence of strains to the intestinal epithelium, pedestal formation and aggregation of polarized actin at sites of bacterial attachment [2].
The genetic determinants for the production of A/E lesions are located on the locus of enterocyte effacement (LEE), a pathogenicity island that contains the genes encoding intimin (eae), a type III secretion system, a number of secreted proteins (ESP) and the translocated intimin receptor (Tir) [2]. Characterization of eae genes revealed the existence of different eae variants. At present, 15 (α1, α2, β1, β2, γ1, γ2/θ, δ/κ, ε, ζ, η, ι, λ, µ, ν, ξ) genetic variants of the eae gene have been identified [3][4][5][6][7]. It is discussed that different intimins may be responsible for different host-and tissue cell tropism [8,9]. In epithelial culture cells EPEC strains produce characteristic adherence patterns: (i) localized adherence (LA), where microcolonies attach after 3 hours incubation to one or two small areas on the cells [10,11]; (ii) diffuse adherence (DA), where bacteria cover the cells uniformly [10]; (iii) enteroadherent-aggregative adherence (AA), where the bacteria have a characteristic stacked-brick-like arrangement [12] and (iiii) localized adherence-like adherence (LAL), characterized by formation of less-compact microcolonies or clusters of bacteria than those by LA [13]. Localized adherence is associated with the presence of the large EPEC adherence factor (EAF) plasmid, on which also the cluster of genes encoding bundle-forming pili (BFP) is present [1]. EPEC are classified as typical, when possessing the EAF plasmid; whereas atypical EPEC strains do not possess the EAF plasmid [14,15].
Typical and atypical EPEC strains usually belong to certain serotype clusters, differ in their adherence patterns on cultured epithelial cells (typical: LA; atypical: DA; AA; LAL), are typically found in different hosts (typical EPEC strains have only been recovered from humans), and display difference in their intimin types [15].
The role and the epidemiology of non-traditional enteropathogenic E. coli serogroup strains in human diarrhea are not well established. E. coli O157:H8 and E. coli O157:H45 EPEC strains have been described to be associ-ated with diarrhea in human [4,16]. Makino et al. [17] reported the first large outbreak with E. coli O157:H45 EPEC in Japan in 1998. Nevertheless, these O157 EPEC strains are not well characterized.
This study reports the first isolation of O157:H45 EPEC strains from cattle and phenotypic and genotypic characteristics of the isolated strains are described.

Results and discussion
All 11 sorbitol-positive E. coli O157:H45 strains, six strains isolated from fattening cattle and five strains isolated from cows tested negative for Shiga toxin genes (stx). The eae gene, which is strongly correlated to the attachingand-effacing lesions, was present in all strains. Therefore, these strains are considered to be EPEC. Our findings emphasize the fact that E. coli with the O157 O antigen are not always STEC but may belong to other pathotypes. Further results of strain characterization are shown in Tab. 2.
In all tested strains, LEE was inserted in selC and thus these strains could be evolutionarily more akin to the EPEC 1 and EHEC 1 clones than to other clones [18]. Further characterization of the eae variants showed in all strains eae subtype α1. Oswald et al. [4] testing in their study the distribution of intimin types among different EPEC strains, found the same eae subtype in O157:H45 strains isolated from human with diarrhea.
The gene encoding the heat-stable enterotoxin (EAST1), representing an additional determinant in the pathogenesis of E. coli diarrhea, was found in ten of the 11 strains. None of the strains, however, carried ehxA genes.
Nine strains were positive for the EAF plasmid and 10 strains harbored the bfpA gene encoding bundlin, the structural subunit of the bundle-forming pilus expressed by typical EPEC strains. The EAF plasmid negative but bfpA positive strain may have a defect in the EAF region that does not interfere with the plasmid's function. All bfpA gene positive strains showed a localized adherence (LA) pattern, the penotpyical characteristic associated with the expression of bundle-forming pili and therefore seem to be a typical EPEC. For HEp-2 and Caco-2 cells similar results were obtained (Figure 1, 2). Caco-2 cells instead of Hela cells were used for adhesion assay and FAS test, because they represent a system close to the human intestinal cells. Furthermore, Vieira et al. [19] have shown that results obtained by Caco-2 cells are comparable to Hela cells.
10 of the 11 strains showed an A/E property with the fluorescent actin staining (FAS) test ( Figure 3). According to Kaper [14] most of our O157:H45 strains have to be classified as typical EPEC strains, because they are possessing the EAF plasmid and are showing the localized adherence (LA) pattern. However, they are harboring astA genes, which is more common for atypical EPEC strains [15].
Further studies are needed to evaluate the role of such EPEC strains isolated from asymptomatic animals.

Conclusions
Cattle have not only to be considered as important asymptomatic carriers of O157 STEC but can also be a reservoir of O157 EPEC strains, which are described in association with human diseases. These strains appear to play a role as food borne pathogens and have to be considered in view of food safety aspects.

Origin of the E. coli strains
The strains characterized in this work were isolated during a recent study on prevalence of E. coli O157 in healthy cat-tle in Switzerland [20]. The study was based on investigations that were carried out within the last one and a half years   Table 1. Differences in the normal PCR mixture and cycling conditions for each PCR were previously described in the cited literature.

Authors' contributions
RS isolated the strains and drafted the manuscript, CZ was responsible for further strain characterization, NB performed adhesion assays and FAS tests, MB performed the PCR typing of eae genes and JE has done serotyping of the strains. All authors read, commented on and approved of the final manuscript.