The purpose of this study was to identify and characterize the development of biofilms of clinical strains of Escherichia coli O157:H7, using different methodologies to evaluate the ability of these strains to form biofilm under different growing conditions and related to the bacterial stress.
Eight clinical strains of E.coli O157:H7 and the reference one were studied to investigate the formation of biofilm in vitro, in glass and plastic tubes and in ELISA?s plates. The latter methodology was optimized doing a kinetic growth at 37ºC in trypticase soy broth, with or without glucose.
The staining of the biofilm was made with crystal violet (CV) and the optical density was determined at 595 nm. The generation of REACTIVE OXYGEN SPECIES (ROS) and the production of NITRIC OXIDE (NO) were measured in the supernatant of cultures grown under aerobiosis and microaerophilic conditions by means of Nitroblue Tetrazolium (NBT) and Griess reaction, respectively.
The biofilm ultrastructure was analysed by CONFOCAL SCANNING LASER MICROSCOPY. In addition, curli expression was studied by congo red, while the cell surface hydrophobicity was determined by partition with xylene.
The strains showed low ability to form biofilm in both glass and plastic tubes. The optimal conditions for the biofilm formation were a dilution 1/10 of the planktonic overnight culture in trypticase soy broth for 48 h. The ability to form biofilm increased with the incubation with glucose. The analysis of the bacterial stress indicated that the incubation with glucose significantly decreased the ROS and NO generation. Similar results were obtained with the cultures grown under aerobiosis and microaerophilic conditions. A low partition was also observed with the organic solvent.
The colonies grown in CONGO RED AGAR showed pink colonies in all strains.
The E.coli O157:H7 strains showed low ability to form biofilm in both, glass and plastic tubes. The absence of black colonies in congo red agar and the lack of hydrophobicity
of bacterial cells indicated to low ability to form biofilm.
The incubation in microaerophilic conditions did not significantly modify the biofilm formation. The addition of glucose decreased the cellular stress of the sesiles cells but increaded the ability of E.coli O157:H7 to form biofilm.
