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)

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].

STEC are responsible for a number of human gastrointestinal diseases, including diarrhea, bloody diarrhea and hemorrhagic colitis (HC). In a proportion of individuals, particularly in children, these conditions may be complicated by neurological and renal sequelae, including hemolytic-uremic syndrome (HUS) [18–20]. Most outbreaks and sporadic cases of HC and HUS have been attributed to O157:H7 STEC strains [20–22]. In contrast, especially in continental Europe, infections by non-O157 STEC, such as O26:H11/H-, O91:H21/H-, O103:H2, O111:H-, O113:H21, O117:H7, O118:H16, O121:H19, O128:H2/H-, O145:H28/H-, and O146:H21, are more common and frequently associated with severe illness in humans [1, 23–27]. Probably, non-O157 STEC also play a more important role in disease in Argentina [28], Australia [29], Chile [1], and South Africa [1]. STEC are characterized by elaborating two groups of potent phage-encoded cytotoxins called Shiga toxins [18, 19]. Pathogenic STEC very often possess other putative virulence factors as intimin (eae) and Enterohemolysin (ehxA) [19, 30, 31].

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.

Of 51 eae positive E. coli strains, 59% were classified as EPEC and 41% as STEC. The serogroups O26, O103, and O145 and serotypes O26:H-, O103:H2, O145:H- were most frequently found. Comparable distributions of EPEC (57%) and STEC (43%) strains and serogroups (44% of strains serotyped as O26, O103, O145, or O156) were reported in strains isolated from healthy cattle in Japan [34]. Of the typical EPEC serogroups (O26, O55, O86, O111, O114, O119, O125, O126, O127, O128, O142, and O156), only six strains were found in the present study: four O26 strains, one O119 strain, and one O128 strain. In bovine STEC strains, only a restricted number of serotypes have been most commonly found [8, 20]: the predominant serotype in the majority of surveys realized in Europe was O113:H21, in America and Australia O26:H11, and in Japan O45:H8/H- and O145:H-. As in the present study, authors in Argentina, Canada, France, Germany, Spain, and the United States have found that many STEC recovered from cattle belonged to serotypes previously associated with human disease [8, 35–38].

Intimin mediates the intimate bacterial attachment to the host cell surface of EPEC and STEC, and is required for the formation of the characteristic A/E lesions. In EPEC and STEC strains isolated from human patients and from healthy infants from Germany and Australia, intimin variants α1, β1, γ1 and γ2/θ were most frequently detected [5, 6, 8, 17, 26]. Moreover, STEC serotypes commonly recovered from outbreaks of HUS and hemorrhagic colitis (O157:H7/H-; O111:H-; O26:H11/H-) typically possess intimin γ1, γ2/θ, and β1 [5, 6, 8, 14].

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 LEE-positive 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.

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. Strains harboring eae-β1 include important human diarrheagenic serotypes such as EPEC O26:H11, O111:H2, O114:H2, O126:H2, O128:H2 and STEC O26:H11/H-, O118:H16 and O177:H- [5, 26, 27], serotypes partly also detected in this study.

Intimin ε1 was described for the first time in human and bovine STEC of serogroup O8, O11, O45, O103, O121, and O165 [5]. In this study, eae-ε1, was detected in three bovine EPEC strains including one O103:H2 strain, and in all 9 bovine STEC O103:H2 strains. Furthermore, in a recent study performed on sheep in Switzerland, intimin ε1 was detected in all STEC O103:H2 [43]. STEC O103:H2 strains have frequently been associated with HUS and hemorrhagic colitis [26, 27, 44, 45].

Intimin γ1 was restricted to STEC and associated with serotypes considered as highly pathogenic to humans: O145:H- (five strains) and, O157:H7 (one strain). In addition to these serotypes, eae-γ1 was previously described in association with STEC O145:H28, and with EPEC O55:H7/H-, from which STEC O157:H7 are believed to have evolved [8, 26, 27, 46].

Interestingly, one bovine EPEC O157:H45 strain harboring eae-α1 was detected. Moreover, eae-α1 was also recently detected in bovine EPEC O157:H45 [33], in two EPEC O142:H6 isolated from monkeys [47] and in a human STEC O177:H7 strain [25]. These findings disagree with the suggested restriction of intimin α1 to human EPEC strains of serotypes O55:H6, O127:H6, H34/H-, O142:H6 [4, 5].

We have identified a new intimin gene (η2) in three bovine bfpA and astA-positive EPEC strains of serotype ONT:H45. The complete nucleotide sequences of the new eae-η2 (AJ879898, AJ879899, AJ879900) variant genes of the three strains were determined. The new intimin η2 also was found in one EPEC ONT:H45 strain isolated from a patient with diarrhea in Spain by Blanco et al. (unpublished data, EMBL nucleotide sequence AJ876652). Recently, a food-borne outbreak of diarrhea involving 41 students (ages 12 to 15) was reported in Japan by Yatsuyanagi et al. [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 ast A 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 stx 2 and eae positive [49–51]. 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 [5–7, 39]. In consequence, apart of the stx 2 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 stx 1, stx 2, eae-γ1, and ehxA, one O26:H- strain harboring stx 2, eae-β1, and ehxA, one O145:H- strain harboring stx 2, eae-γ1, and ehxA, and one O157:H7 strain harboring stx 2, eae-γ1, and ehxA.

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.