DIFFERENTIAL ADHERENCE OF LOCUS OF ENTEROCYTE EFFACEMENT-NEGATIVE SHIGA-TOXIN PRODUCING Escherichia coli STRAINS HARBORING lpfAO113 AND/OR iha GENES to HEp-2 CELLS

GALLI L; LARZÁBAL M; LEOTTA GA; MERCADO EC; RIVAS M

Buenos Aires, Argentina

Simposio; 7th International Symposium on Shiga Toxin (verocytotoxin)- producing Escherichi coli infections; 2009

Shiga toxin-producing Escherichia coli (STEC) strains that do not carry the locus of enterocyte effacement (LEE) are commonly isolated from sporadic cases and small outbreaks, although little is known about the way in which these organisms adhere to and colonize the human intestine. In this work we compared the adherence phenotype of LEE-negative STEC isolates, which were previously characterized as carrying the lpfAO113 and/or iha genes. Three lpfAO113 positive bovine isolates belonged to serotypes O136:H12, O7:H- and ONT:H-, respectively. Two human strains harboring the genes iha or iha/lpfAO113 corresponded to serotypes ONT:H4 and O143:H-, respectively. All the strains were negative for the putative adherence factors saa, efa1, lpfAO157/OI-154, lpfAO157/OI-141 and toxB. Qualitative and quantitative assessment of bacterial adherence to HEp-2 cells was determined by Giemsa staining and bacterial enumeration. Preliminary assays showed that lpfAO113 positive strains exhibited an increased adherence phenotype when they were grown at 37 ºC overnight in LB broth (pH 7) in static conditions and were inoculated at a multiplicity of infection of 25:1. Semiconfluent HEp-2 cells on glass coverslips were incubated, in the presence of 1% D-mannose, with 107 bacteria/well, for 1.5 h at 37 ºC and 5% CO2, washed with PBS, and incubated an additional period of 4 h. To quantify adherence, cells were washed, lysed with 1% Triton X-100 and resuspended in LB for quantification by plating serial dilutions. E. coli DH5α lacking iha/lpfAO113 genes was used as negative control. Ability of the strains to invade HEp-2 cells was compared to those of the reference strain EIEC C481. The total number of cell-associated bacteria was determined as above. To obtain the number of intracellular bacteria, a second set of infected wells was washed and incubated with 100 µg/ml gentamicin for 90 min. Then, cells were washed, lysed with 1% Triton X-100 and resuspended in LB for quantification by plating serial dilutions. Experiments were performed three times, each time in duplicate. Statistical differences in adherence and invasion were determined by one-way analysis of variance. All the lpfAO113+ strains aggregated on the cell surface, in a localized-like or diffuse adherence pattern, while the iha+ strain showed a detachment adherence pattern. Quantitative assays showed that the STEC ONT:H4/iha+ strain, was significantly less adherent to HEp-2 cells than the lpfAO113+ isolates (P=0.0009). Invasion assays demonstrated that lpfAO113+ strains invaded HEp-2 cells, but the iha+ and iha/lpfAO113+ strains were not internalized (P=0.00001). In this study, we corroborated that some LEE-negative STEC strains may employ a host cell invasion mechanism for pathogenesis that could compensate the absence of LEE. It is the first time that stx1strains from bovine origin belonging to serotypes O136:H12, O7:H- and ONT:H-, were described as invasive to HEp-2 cells. Our finding shows that the LPF operon is widely distributed in LEE-negative STEC strains from different origin.