Typing of Intimin Genes in Human and Animal Enterohemorrhagic and EnteropathogenicEscherichia coli: Characterization of a New Intimin Variant

Abstract

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) produce the characteristic "attaching and effacing" (A/E) lesion of the brush border. Intimin, an outer membrane protein encoded by eae, is responsible for the tight association of both pathogens with the host cell. Several eae have been cloned from different EPEC and EHEC strains isolated from humans and animals. These sequences are conserved in the N-terminal region but highly variable in the last C-terminal 280 amino acids (aa), where the cell binding activity is localized. Based on these considerations, we developed a panel of specific primers to investigate the eae heterogeneity of the variable 3 region by using PCR amplification. We then investigated the distribution of the known intimin types in a large collection of EPEC and EHEC strains isolated from humans and different animal species. The existence of a yet-unknown family of intimin was suspected because several EHEC strains, isolated from human and cattle, did not react with any of the specific primer pairs, although these strains were eae positive when primers amplifying the conserved 5 end were used. We then cloned and sequenced the eae present in one of these strains (EHEC of serotype O103:H2) and subsequently designed a PCR primer that recognizes in a specific manner the variable 3 region of this new intimin type. This intimin, referred to as "," was present in human and bovine EHEC strains of serogroups O8, O11, O45, O103, O121, and O165. Intimin is the largest intimin cloned to date (948 aa) and shares the greatest overall sequence identity with intimin ␤, although analysis of the last C-terminal 280 aa suggests a greater similarity with intimins ␣ and ␥.

Isolation from a sheep of an attaching and effacing Escherichia coli O115:H− with a novel combination of virulence factors

“…This intimin subtype has been identified among several STEC serotypes of humans and cattle, with the majority (19 of 25) being serogroup O103 [20]. Only 2 of the 25 å intimin isolates (both O103:Hnd) were from non-human (cattle) sources [20]. In man, the EHEC O103:H2 serotype has been associated with HUS in Europe [42][43][44], the USA [45] and Canada [46], although it is only rarely associated with diarrhoeagenic disease in animals [47].…”

“…To date, at least five different intimin subtypes have been described from EPEC and EHEC isolates [20] and their distribution may account for the ability of the intimin-producing strains to colonise different tissues and hosts. The å intimin subtype is the most recent form of the eaeA gene to be described [20].…”

“…The å intimin subtype is the most recent form of the eaeA gene to be described [20]. This intimin subtype has been identified among several STEC serotypes of humans and cattle, with the majority (19 of 25) being serogroup O103 [20].…”

“…The PCR primers used in this study are defined in Table 2 [13,[20][21][22][23]. Template DNA was purified from bacterial strains by phenol/chloroform extractions.…”

Genetic Characteristics and Antimicrobial Resistance of Escherichia Coli From Japanese Macaques (Macaca Fuscata) in Rural Japan

“…DNA extracted with a DNeasy TM Tissue Kit (QIAGEN, Hilden, Germany), or boiled enrichment culture was used as templates. Oligonucleotide primers (Table 2) for PCR were as follows: SK1, SK2 for eae (Oswald et al, 2000), Vtcom-u, Vtcomd for stx (Yamasaki et al, 1996), LT L , LT R for elt (Toma et al, 2003), and AL65, AL125c for est (Toma et al, 2003). PCR was performed under the following conditions: initial denaturation for 5 min at 94 C, 30 cycles of 1 min at 94 C, 1 min at 55 C, 1 min at 72 C, and a final extension step of 7 min at 72 C. PCR amplicons were electrophoresed on 2% SeaKem GTG agarose gel (Takara, Otsu, Shiga, Japan) in 13 TAE buffer.…”