Adherence patterns of diarrheagenic Escherichia coli strains isolated from children with gastrointestinal diarrhea and matched controls in a Hep-2 cell adhesion assay

Escherichia coli is considered as the main cause of epidemic outbreaks and endemic cases of child diarrhea. In the present study, the capacity of a large collection of Escherichia coli (E. coli) strains and matched controls was investigated to determine their adherence pattern on Hep-2 cell lines. A total of 66 pure E. coli strains, which were previously isolated from children aged less than 12 years with diarrhea, were selected for adherence assay. Moreover, 2 E. coli strains, with known adherence patterns, obtained from the Persian Type Culture Collection were used as the control. Hep-2 cell monolayers (50 to 70% confluence) were grown on glass cover slips. Subsequently, 20 μl of bacterial cultures (2 × 106 cfu/ml bacteria) was added into each well, and the plates were incubated in a humid atmosphere with 5% CO2 for 3 hours. Then, culture medium was aspirated from the monolayers, which were washed 4 times with phosphate-buffered saline, fixed in 70% aqueous methanol for 5 minutes, stained with Giemsa stain, and examined using light microscopy. In total, 66 E. coli strains were evaluated for their adherence pattern on Hep-2 cell line. All the isolates adhered to the Hep-2 cells with the exception of 3. The other 63 showed aggregative adherence (AA) (34%), localized adherence (LA) (31%), or diffuse adherence (DA) (29%) patterns. The present investigation revealed that this assay is convenient and economical for identification of a large number of diarrheagenic E. coli isolates’ adherence to Hep-2 cell lines.