Determination of adhesin gene sequences in, and biofilm formation by, O157 and non-O157 Shiga toxin-producing Escherichia coli strains isolated from different sources

Abstract

Biofilm formation by Shiga toxin-producing Escherichia coli (STEC) has been associated with the expression of different adhesins (type 1 fimbria, curli, Ag43, Cah, and EhaA). In this study, biofilm formation and the presence of adhesin-related gene sequences were determined by PCR in 18 O157 strains and 33 non-O157 strains isolated from different sources (human, animal, food, and water). The expression of different adhesins was also assessed by reverse transcription-PCR (RT-PCR), Congo red agar plates, and mannose-sensitive hemagglutination (MSHA) assay. Biofilm formation occurred in 5/18 (28%) O157 STEC strains and 17/33 (51%) non-O157 STEC strains from different serotypes and sources, when the assays were performed at 28°C for 48 h. Among the non-O157 biofilm-producing isolates, 12/17 (71%) expressed type 1 fimbriae and 11/17 (65%) expressed curli and produced cellulose, while 8/17 (47%) were considered to be Ag43(+) by RT-PCR. Among O157 strains, a close correlation was observed between biofilm formation and expression of curli and cellulose. In non-O157 strains, it seems that, in addition to the presence of curli, the ability to form biofilm is associated with the presence of other factors such as type 1 fimbriae and autotransporter proteins, which may contribute to the persistence of these organisms in the environment.

Trial registration: ClinicalTrials.gov NCT00464308.

Figures

FIG. 1.

Representative RT-PCR analysis for identification of Ag43 (A), Cah (B), and EhaA (C) autotransporter proteins from E. coli strains. Lanes 1 in panels A to C contained a 100-bp molecular size standard. (A) Lanes: 2, cDNA from strain 243/01 (O79:H14); 3, cDNA from strain 473/01 (O105:H18); 4, digested RNA from strain 243/01 (O79:H14). (B) Lanes: 2, cDNA from strain B1/1 (O157:H7); 3, digested RNA from strain B1/1. (C) Lanes: 2, cDNA from strain 463/01 (O91:H21); 3, cDNA from strain 473/01 (O105:H18); 4, digested RNA from strain 473/01 (O105:H18).

FIG. 2.

Negative staining (NS) and immunogold labeling (IGL) of representative O157 and non-O157 STEC pair of strains previously detected as curli− and curli+ after growth on CFA agar containing Congo red dye at 28°C for 48 h. (A to D) The absence of fimbrial structures in curli− variants (A and B) and the presence of hairlike fimbrial structures in curli+ variants (C and D) can be observed. (E and F) IGL labeling using anticurli antibody and anti-rabbit antibody labeled with 10-nm colloidal gold confirm the presence of curli fimbriae. Bars, 0.5 μm.

FIG. 3.

Quantitative comparison of biofilm formation by pairs of curli+ and curli− variants of non-O157 (A) and O157 (B) STEC strains in polystyrene plates after growth in LB broth at 28°C for 48 h. Biofilm assays were performed on pairs of curli+ (+) and curli− (−) variants of E. coli strains as described in Materials and Methods, and each bar represents the mean absorbance value at 540 nm plus standard deviation (error bar) for three independent experiments. Values for the biofilm-forming strains that were significantly different (P < 0.05, Student's t test) from the values for negative-control E. coli HB101 are indicated by an asterisk. (C) Light microscopy of adherence of a curli+ O157 STEC strain to polystyrene plates at 28°C (×1,000). Adherence starts after 2 h of incubation and gradually increases until 48 h.