Serotypes, intimin variants and other virulence factors of eae positive Escherichia coli strains isolated from healthy cattle in Switzerland. Identification of a new intimin variant gene (eae-η2)

Background Enteropathogenic Escherichia coli (EPEC) and Shigatoxin-producing Escherichia coli (STEC) share the ability to introduce attaching-and-effacing (A/E) lesions on intestinal cells. The genetic determinants for the production of A/E lesions are located on the locus of enterocyte effacement (LEE), a pathogenicity island that also contains the genes encoding intimin (eae). This study reports information on the occurrence of eae positive E. coli carried by healthy cattle at the point of slaughter, and on serotypes, intimin variants, and further virulence factors of isolated EPEC and STEC strains. Results Of 51 eae positive bovine E. coli strains, 59% were classified as EPEC and 41% as STEC. EPEC strains belonged to 18 O:H serotypes, six strains to typical EPEC serogroups. EPEC strains harbored a variety of intimin variants with eae-β1 being most frequently found. Moreover, nine EPEC strains harbored astA (EAST1), seven bfpA (bundlin), and only one strain was positive for the EAF plasmid. We have identified a new intimin gene (η2) in three bovine bfpA and astA-positive EPEC strains of serotype ONT:H45. STEC strains belonged to seven O:H serotypes with one serotype (O103:H2) accounting for 48% of the strains. The majority of bovine STEC strains (90%) belonged to five serotypes previously reported in association with hemolytic uremic syndrom (HUS), including one O157:H7 STEC strain. STEC strains harbored four intimin variants with eae-ε1 and eae-γ1 being most frequently found. Moreover, the majority of STEC strains carried only stx1 genes (13 strains), and was positive for ehxA (18 strains) encoding for Enterohemolysin. Four STEC strains showed a virulence pattern characteristic of highly virulent human strains (stx2 and eae positive). Conclusion Our data confirm that ruminants are an important source of serologically and genetically diverse intimin-harboring E. coli strains. Moreover, cattle have not only to be considered as important asymptomatic carriers of O157 STEC but can also be a reservoir of EPEC and eae positive non-O157 STEC, which are described in association with human diseases.


Background
Enteropathogenic Escherichia coli (EPEC) and Shiga toxinproducing Escherichia coli (STEC) represent two of the at least six different categories of diarrheagenic E. coli recognized at present [1]. Unlike other diarrheagenic E. coli, EPEC and STEC share the ability to introduce attachingand-effacing (A/E) lesions on intestinal epithelial cells. A/ E lesions are characterized by destruction of the microvillus brush border through restructuring of the underlying cytoskeleton by signal transduction between bacterial and host cells, intimate adherence of strains to the intestinal epithelium, pedestal formation and aggregation of polymerized actin at the sites of bacterial attachment [1,2]. The genetic determinants for the production of A/E lesions are encoded in a pathogenicity island called the locus of enterocyte effacement (LEE) [3]. The LEE encodes the outer membrane protein intimin, which is encoded by the eae gene (for E. coli attachment effacement) localized in the central region of LEE, a type III secretion system, a number of secreted proteins (ESP), and Tir (for translocated intimin receptor), a protein encoded upstream of the eae gene, which is translocated into host cells [2]. Characterization of eae genes revealed the existence of different variants. At present, 17 genetic variants (α1, α2, β1, ξR/β2B, δ/κ/β2O, γ1, θ/γ2, ε1, νR/ε2, ζ, η, ι1, µR/ι2, λ, µB, νB, ξB) have been identified (Table 4) [4][5][6][7][8][9]. The heterogeneous C-terminal (3') end of intimin is responsible for receptor binding, and it is discussed that different intimin variants may be responsible for different host and tissue cell tropism [6,[10][11][12]. Adu-Bobie et al. [4] found that antigenic variation exists within the cell-binding domain of intimins expressed by different clinical human EPEC and STEC isolates and defined four intimin types (α, β, γ, δ) based on type-specific PCR assays that used oligonucleotide primers complementary to the 3'end of specific eae genes. Oswald et al. [5] described a type-specific PCR assay which identifies a fifth intimin variant (intimin ε; referred in the present study as ε1). They divided the intimin alleles α, β, and γ based on restriction fragment length polymorphism (RFLP) analysis of PCR products into α1, α2, β1, β2, γ1 and γ2 subtypes. Furthermore, Oswald et al. [5] reclassified the δ type of Adu-Bobie et al. [4] as a β2 subtype (referred in the present study as d/ β2O). Tarr and Whitam [13] presented a paper on the molecular evolution of intimin genes in human STEC and EPEC O111 clones and found two new types (ζ and θ). The new ζ intimin type also was identified by Jores et al [11] in bovine STEC and Blanco et al. [7] in ovine STEC. Zhang et al. [6] observed that the sequences of eae-γ2 and eae-θ were almost identical (99%), and they believe that these two sequences should be considered one eae variant (γ2/θ), as is referred in the present study. Recently, Zhang et al. [6] determined the sequences of three new intimin variant genes (κ, η, and ι; intimin ι referred in the present study as ι1) found in human STEC strains. The sequence of the 3'variable region of eae gene of a new intimin (λ) has been submitted to GenBank by China (unpublished data) (Accession no. AF439538) and the whole intimin λ gene was sequenced by Blanco et al. (unpublished data) from a human strain (Accession no. AJ715409) and Ramachandran et al. [14] (Accession no. AF530557) from a bovine strain. Ramachandran et al. [14] identified three new intimin genes in ruminant E. coli strains belonging to serotypes ONT:H-(intimin µ, referred in the present study as µR/ι2), O2-related:H19 (intimin ν, referred in the present study as νR/ε2) and ONT:HNT (intimin ξ, referred in the present study as ξR/β2B) and they have also developed an intimin typing PCR-RFLP scheme that reliably differentiates 14 intimin variants. When Ramachandran et al. [14] submitted to GenBank the nucleotide sequences of µ, ν, and ξ genes these intimin types were designated ι2, ε2 and β2, respectively. Blanco et al. [8] have identified four new intimin variant genes that they originally designated as β2, µ, ν, and ξ when the sequences were submitted to EMBL Nucleotide Sequence Database, and before knowing the results obtained by Ramachandran et al. [14]. The intimin β2 found by Blanco et al. (unpublished data) in human typical EPEC strains of classical EPEC serotype O119:H6 is identical to intimin ξ described by Ramachandran et al. [14] in one bovine strain of serotype ONT:HNT. Thus, in this study our β2 intimin is referred as ξR/β2B. The other three intimins (µ, ν, and ξ) discovered by Blanco et al. [[8], unpublished data] are different to the existing intimin types and are referred to as µB, νB, and ξB in this study, respectively. EPEC strains are defined as eae-haboring diarrheagenic E. coli that possess the ability to form A/E lesions on intestinal cells and that do not possess Shiga toxin genes [15]. Most EPEC strains belong to a series of O antigenic groups known as EPEC serogroups: O26, O55, O86, O111, O114, O119, O125, O126, O127, O128, O142, and O158. EPEC are further classified as typical, when possessing the EAF (for EPEC adherence factor) plasmid that encodes localized adherence (LA) on cultured epithelial cells mediated by the Bundle Forming Pilus (BFP); whereas atypical EPEC strains do not possess the EAF plasmid [15,16]. Typical EPEC, a major cause of infant diarrhea in developing countries, are rare in industrialized countries, where atypical EPEC seem to be a more important cause of diarrhea [1,16]. Typical and atypical EPEC strains usually belong to certain serotype clusters, differ in their adherence patterns on cultured epithelial cells (typical: LA; atypical: diffuse adherence (DA); aggregative adherence (AA); localized adherence-like adherence (LAL)), are typically found in different hosts (typical EPEC strains have only been recovered from humans), and display differences in their intimin variants [16]. Atypical EPEC appear to be more closely related to STEC and as such are considered emerging pathogens [16,17].
Although a number of studies have determined the eae subtypes of STEC strains isolated from humans, only a limited number of studies have been undertaken to determine the intimin variants from a diverse collection of STEC and EPEC isolated from cattle. Ruminants, especially cattle, represent an important reservoir of atypical EPEC and STEC [16,20,32,33]. This study reports information on the occurrence of eae positive E. coli carried by healthy cattle at the point of slaughter, and on serotypes, intimin variants, and further virulence factors of isolated EPEC and STEC strains.

Detection and isolation of eae positive E. coli strains
Of the 330 fecal samples collected from cattle at slaughter, 132 (40%) tested positive for eae genes. From 51 randomly selected eae PCR-positive samples, 51 eae positive E. coli strains were isolated by colony dot blot hybridization, confirmed as E. coli by biochemical properties, and further characterized by phenotypic and genotypic traits. Thirty strains carried no stx genes and were therefore classified as EPEC, whereas the remaining 21 strains possessed at least one Shiga toxin gene, and were therefore considered as STEC.

Identification of a new intimin variant gene (eae-η2). Sequence comparison
The complete nucleotide sequence of the new η2 variant gene in three bovine bfpA and astA-positive EPEC strains of serotype ONT:H45 was determined. Furthermore, a fragment (566 bp strain FV5126 to 1728 bp strain FV5114/2) of the 3'variable region of the eae gene of seven representative strains was amplified and sequenced. The eae sequences were deposited in the European Bioinformatics Institute (EMBL Nucleotide Sequence Database) and the accession numbers assigned are indicated in table 3.

Further characterization of EPEC and STEC strains
Nine EPEC strains harbored the astA gene encoding for EAST1 (Table 1). Additionally, three astA positive strains of serotype ONT:H45 harbored the bfpA gene encoding bundlin, whereas only one strain was positive for the EAF plasmid. Overall, 23 different associations of serotypes with virulence factors were identified in EPEC strains.
Of the 21 eae positive E. coli harboring genes encoding for Shiga toxins, the majority (13 strains) carried only stx1 genes, 4 strains only stx2 genes, and 4 strains both toxin genes ( Table 2). The only stx1 positive strains belonged to serotypes O5:H-(2 strains), O103:H2 (10 strains) and O111:H21 (1 strain), the only stx2 positive strains to serotypes O26:H-, O26:H2, O145:H-, and O157:H7, and all the strains positive for both toxin genes to serotype O145:H-. Moreover, the majority of STEC strains (86%) tested positive for the ehxA gene encoding for Enterohemolysin. Overall, ten different associations of serotypes with virulence factors were identified.  Overall, the 51 isolated eae positive bovine E. coli strains comprised a variety of 10 intimin variants, and most of them harbored eae-β1(33%), eae-ε1 (26%) and eae-γ1(16%). As it is known that different intimin variants are associated with distinctive phylogenetic lineages of LEEpositive E. coli [4,39,40], the identified variety of bovine eae positive strains further substantiates the reservoir function of cattle for infections of humans with LEE positive strains.

Discussion
Intimin β1 appears to be the most widespread variant [7,8,14,27,39,41,42]. In that it has been found in both EPEC and STEC strains from humans and several animal species, and its presence is associated with multiple E. coli serotypes.  [48]. The implicated organism was a EPEC ONT:H45, which hybridized with the probes for eaeA, astA and bfpA genes.
In our study, about one third of EPEC strains harbored astA as further putative virulence factor; whereas only seven EPEC strains harbored bfpA encoding bundling, the structural subunit of the bundle-forming pilus in typical EPEC strains, and only one EPEC of serotype O157:H45 was positive for the EAF plasmid. Overall, bfpA seems to be isolated very rarely from bovine EPEC strains, since there are scarcely any reports of E. coli strains with bfpA isolated from cattle. However, in a recent study, we characterized 11 O157:H45 EPEC strains isolated from cattle in Switzerland and found 10 bfpA positive strains among them [33].
To assess the pathogenicity of STEC, further evaluation of virulence factors in addition to serotype is necessary. O157 and non-O157 STEC strains isolated from patients with severe symptoms such as bloody diarrhea, HC, and HUS frequently show a typical virulence spectrum, with such strains tending to be stx2 and eae positive [49][50][51]. Moreover, it was previously shown that bovine and human strains harboring the eae gene were statistically more likely to be positive for the ehxA gene encoding for Enterohemolysin [7,26,52]. However, the impact of Enterohemolysin, which was found in the majority of STEC strains, is controversially discussed. Additionally, STEC commonly recovered from outbreaks of HUS and HC typically possessed eae-β1, eae-γ1 and γ2/θ [5][6][7]39]. In consequence, apart of the stx2 gene, which was previously reported to correlate with severe disease in humans, intimin subtyping may facilitate further understanding of associations among serotype, eae and stx subtype. In consequence, seven STEC strains (14% of all eae positive strains and 33% of STEC strains) showed a virulence pattern characteristic of highly virulent human strains: four O145:H-strains harboring stx1, stx2, eae-γ1, and ehxA, one O26:H-strain harboring stx2, eae-β1, and ehxA, one O145:H-strain harboring stx2, eae-γ1, and ehxA, and one O157:H7 strain harboring stx2, eae-γ1, and ehxA.

Conclusion
Our data confirm that ruminants are an important source of serologically and genetically diverse intimin-harboring E. coli strains. Moreover, cattle have not only to be considered as important asymptomatic carriers of O157 STEC but can also be a reservoir of EPEC and eae positive non-O157 STEC, which are described in association with human diseases. The fecal carriage of foodborne pathogens among livestock animals at slaughter is strongly correlated with the hazard of carcasses contamination. In order to reduce the risk represented by STEC and EPEC, the maintenance of slaughter hygiene is consequently of central importance in meat production.

E. coli strains
From 330 fecal samples, collected from Swiss cattle at slaughter, 10 g were each enriched in 100 ml brilliant green bile broth (BBL, Cockeysville, Md.) at 37°C for 24 h. The enriched samples were streaked onto sheep blood agar (Difco Laboratories, Detroit, Mich.; 5% sheep blood Oxoid, Hampshire, UK), and after incubation at 37°C for another 24 h, the colonies were washed off with 2 ml of 0.85% saline solution. Two µl of each plate eluate were then evaluated by PCR with primers EAE-1 and EAE-2 (Table 5) targeting sequences at the 5' eae conserved region detecting all types of eae described at the moment. From the 132 eae PCR-positive samples, 51 eae PCR-positive samples were then randomly selected for strain isolation with an eae DNA probe and colony dot-blot hybridization.
The eae probes were prepared by labeling eae-PCR amplicons from E. coli O157:H7 strain 857/03 with DIG High Prime kit (Roche, Mannheim, Germany). Briefly, for colony hybridization, the 51 eae positive samples were plated onto sheep blood agar and incubated overnight at 37°C. Colonies were transferred to a nylon membrane (Roche), and lysed following standard methods. After washing, crosslinking, and prehybridization in DIG-Easy-Hyb buffer (Roche) at 37°C for about 30 min, hybridization of membranes with eae DNA probes was performed overnight at 42°C. After washing in pre-heated primary and secondary wash buffers, the presence of labeled probe was detected with in alkaline phosphatase-conjugated antibody detection kit and NBT/BCIP stock solution according to the instructions of the manufacturer (Roche). Positive colonies were picked from the original sheep blood agar and confirmed as E. coli by biochemical properties (acid production from mannitol, the o-nitrophenylβ-D-galactopyranoside (ONPG) test, H 2 S and indole production, and proof of urease and lysine decarboxylase activities) and by PCR to be eae positive [7]. One randomly chosen colony per sample was used for further strain characterization.  Tables 5 and 6. Differences in the normal PCR mixture and cycling conditions for each PCR were previously described in the cited literature. To identify eae variants, intimin type specific PCR assays using primers complementary to the heterogeneous 3' end of intimin genes were performed [8,9]. For detection of further putative virulence genes all strains were examined for the presence of stx genes with primers VT1 and VT2 [54]. Stx positive strains were examined for the presence of stx1 and stx2 genes [55,56]. Stx negative strains were further examined for the presence of the EAF plasmid [57], the bfpA gene on the EAF plasmid [58], and the astA gene encoding EAST1 [59].

Sequencing of the intimin (eae) genes
The nucleotide sequence of the amplification products purified with a QIAquick DNA purification kit (Qiagen) was determined by the dideoxynucleotide triphosphate chain termination method of Sanger, with the BigDye Terminator v3.1 Cycle Sequencing Kit and an ABI 3100 Genetic Analyzer (Applied Bio-Systems).

Phylogenetic analyses
Genetic distances and phylogenetic trees of eae sequences were calculated and constructed with the CLUSTAL W program [60]included in the EMBL sofware http:// www.ebi.ac.uk/clustalw/.
Sequence Database) and the accession numbers assigned are indicated in Table 3.