First isolation and further characterization of enteropathogenic Escherichia coli (EPEC) O157:H45 strains from cattle
© Stephan et al; licensee BioMed Central Ltd. 2004
Received: 16 January 2004
Accepted: 17 March 2004
Published: 17 March 2004
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, ehx A, ast A, 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.
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.
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.
Enteropathogenic Escherichia coli (EPEC), mainly causing infantile diarrhoea, represents one of the at least six different categories of diarrheagenic E. coli with corresponding distinct pathogenic schemes . Most of the EPEC strains belong to a series of O antigenic groups known as EPEC O serogroups: O26, O55, O86, O111, O114, O119, O125, O126, O127, O128, O142 and O158.
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 .
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) . 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 [3–7]. 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 ; (iii) enteroadherent-aggregative adherence (AA), where the bacteria have a characteristic stacked-brick-like arrangement  and (iiii) localized adherence-like adherence (LAL), characterized by formation of less-compact microcolonies or clusters of bacteria than those by LA . 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 . 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 .
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 associated with diarrhea in human [4, 16]. Makino et al.  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
Sequences and predicted lengths of PCR amplification products of oligonucleotide primers used
Oligonucleotide sequence (5'-3')Sequence
CCA TCA ACA CAG TAT ATC CGA
GGT CGC GAG TGA CGG CTT TGT
CAG GGT AAA AGA AAG ATG ATA A
TAT GGG GAC CAT GTA TTA TCA
AAT GGT GCT TGC GCT TGC TGC
GCC GCT TTA TCC AAC CTG GTA
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 . Further characterization of the eae variants showed in all strains eae subtype α1. Oswald et al.  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 ehx A genes.
According to Kaper  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 .
Further studies are needed to evaluate the role of such EPEC strains isolated from asymptomatic animals.
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 cattle in Switzerland . The study was based on investigations that were carried out within the last one and a half years (January 2002 – June 2003) in two EU-approved slaughterhouses. From 2,930 bovine fecal samples (1,183 calves, 1,201 fattening cattle and 546 cows), 11 sorbitol-fermenting E. coli O157:H45 strains (from 6 fattening cattle and 5 cows) were isolated. The determination of the O and H antigens was performed as previously described using all available O (O1 to O181) and H (H1 to H56) antisera .
Genotypic characterization of the O157:H45 strains
Characterization results of 11 sorbitol-fermenting O157:H45 EPEC strains isolated from cattle in Switzerland
No. of strains
LEE in selC
To verify whether LEE was inserted downstream of the selC locus, PCR reactions that amplify the junctions of this locus with the E. coli chromosome were performed according to Sperandio et al. . Primers K255 (5'-GGTTGAGTCGATTGATCTCTGG-3') and K260 (5'-GAGCGAATATTCCGATATCTGGTT-3') were used for the right junction; K296 (5'-CATTCTGAAACAAACTGCTC-3') and K295 (5'-CGCCGATTTTTCTTAGCCCA-3') for the left junction, and K261 (5'-CCTGCAAATAAACACGGCGCAT-3') and K260 for the intact selC gene.
Adhesion assay and FAS test
E. coli colonies were characterized by the pattern of adherence to HEp-2 and Caco-2 cells as described by Karch et al.  with minor modifications. Briefly, HEp-2 and Caco-2 cells were grown for 48 h with 5% CO2 on sterilized coverslips (13 mm diameter; Bibby Sterilin™ Ltd., Stone, England) in 24-well flat-bottom tissue culture plates with low evaporation lid (16 mm diameter; BD Falcon™, Bedford, USA) containing Minimum essential medium (MEM with Earle's salts, 25 mM HEPES, without L-Glutamine;GIBCO™ Invitrogen AG, Basel, Switzerland) supplemented with fetal calf serum (FCS, 10%, Bio Concept™, Allschwil, Switzerland), MEM Non Essential Amino Acids (MEM NEAA (100x) without L-Glutamine, GIBCO™ Invitrogen AG, Basel, Switzerland) and GlutaMAX™I Supplement 200 mM (100x), (GIBCO™ Invitrogen AG, Basel, Switzerland). Bacteria used for the adhesion assay were grown over night in LB-broth Miller (Becton Dickinson, Maryland, USA). Prior to incubation, the confluent cells were washed two times with Dulbecco's phospate buffered saline (D-PBS (1x) without Calcium and Magnesium, GIBCO™ Invitrogen AG, Basel, Switzerland). E. coli diluted 1:100 in 1 ml fresh media (MEM with supplements) was added to the cells and was incubated for 3 h at 37°C and 5% CO2. After this incubation period, monolayers were washed five times with D-PBS (3 h assay) or monolayers were washed ten times with D-PBS, fresh media (MEM with supplements) was added and an additional incubation period of 3 h proceeded (6h assay). Subsequently, the monolayers were washed five times with D-PBS and the cells were fixed for 10 min with 4 Methanol. After the staining with May-Grünwald-Giemsa (Fluka™, Buchs SG, Switzerland), the coverslips were examined by light microscopy to determine the adherence patterns of the strains.
The following fluorescent actin-staining (FAS) test was performed on Caco-2 cells according to Karch et al.  with minor modifications. The cells were fixed with 4% formaldehyde in D-PBS, then washed three times with D-PBS and incubated for 5 min with 0.1% Triton X-100 (Fluka™, Buchs SG, Switzerland) in D-PBS. After another three washes with D-PBS, the monolayers were incubated with 5 μg/ml of fluorescein isothiocyanate labelled phaloidin (Sigma, Steinheim, Germany) for 30 min. After another three washes with D-PBS, the coverslips were examined by fluorescence microscopy (Leica ™).
We thank Sandra Schumacher and Carmen Kaiser for her excellent technical assistance. This study was partly supported by a grant from the Fondo de Investigación Sanitaria (FIS G03/025-COLIRED-O157).
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