HEp-2 cell adherence, actin aggregation, and intimin types of attaching and effacing Escherichia coli strains isolated from healthy infants in Germany and Australia

Abstract

Fecal samples from healthy children under 2 years of age living in Berlin, Germany (205 infants), and Melbourne, Australia (184 infants), were investigated for the presence of attaching and effacing (AE) Escherichia coli (AEEC) strains by screening for eae (intimin) genes. Twenty-seven AEEC strains were isolated from 14 children (7.6%) from Melbourne and from 12 children (5.9%) from Berlin. The 27 AEEC strains were classified as enterohemorrhagic E. coli (one strain, producing Shiga toxin 1), typical enteropathogenic E. coli (EPEC) (one strain carrying an EPEC adherence factor [EAF] plasmid), and atypical EPEC (25 strains negative for Shiga toxins and EAF plasmids). The AEEC were divided into 18 different serotypes, O-nontypeable and O-rough strains. Typing of their intimin genes revealed the presence of intimin alpha in 6 strains, intimin beta in 11 strains, intimin gamma in 7 strains, intimin zeta in 2 strains, and intimin eta in one strain. Analysis of HEp-2 cell adherence showed diffuse adherence or localized adherence-like patterns in 26 AEEC strains; local adherence was found only with the EAF-positive strain. Ten AEEC strains showed an AE property with the fluorescent actin staining (FAS) test. The introduction of an EAF plasmid (pMAR7) converted 11 FAS-negative AEEC strains to FAS positive and increased the FAS reaction in six FAS-positive AEEC strains, indicating that the genes needed for the AE phenotype were functional in these strains. Our finding indicates that atypical EPEC strains could play a double role as strains that naturally immunize against intimin in humans and as reservoirs for new emerging human pathogenic EPEC strains.

FIG. 1.

(A) Plasmid DNA patterns of AEEC and their pMAR7-carrying derivative strains separated on a 0.7% agarose gel. Strain MG1655 (E. coli K-12) served as the plasmid-free control strain. Lanes: 1, MG1655 (pMAR7); 2, MG1655; 3, LTEC93034; 4, LTEC93034 (pMAR7); 5, KK130/1; 6, KK130/1 (pMAR7); 7, KK122/9; 8, KK122/9 (pMAR7); 9, F91/1429-2; 10, F91/1429-2 (pMAR7); 11, F91/3743-2; 12, F91/3743-2 (pMAR7); 13, LTEC94460; 14, LTEC94460 (pMAR7); 15, MG1655 (pMAR7). (B) Southern blot of the agarose gel shown in panel A after hybridization with the EAF probe. A hybridization signal was obtained only with pMAR7 derivative strains. Multimeric forms of pMAR7 leading to two hybridization signals with the EAF probe were found in preparations from some of the strains (lanes 6, 8, and 14). The position of pMAR7 is indicated on both sides of the figure.

FIG. 2.

HindIII-digested plasmid DNA of AEEC and pMAR7-carrying derivative strains after gel electrophoresis on 1% agarose. Lanes: M, molecular weight standard (λ DNA/HindIII fragments and φX 174 RF DNA, HaeIII fragments (Invitrogen); 1, chromosomal DNA (MG1655); 2, MG1655 (pMAR7); 3, KK122/9; 4, KK122/9 (pMAR7); 5, KK125/5; 6, KK125/5 (pMAR7); 7, KK130/1; 8, KK130/1 (pMAR7).