Quantitative analysis of Staphylococcus epidermidis biofilm on the surface of biomaterial

Staphylococcus epidermidis biofilm is considered to be an important cause of device-related infection. Polysaccharide intercellular adhesin (PIA), encoded by the icaADBC locus, has been found to be a functional component of S. epidermidis biofilm, but the sequential change of the ica gene expression during biofilm development is still unclear. We have established a quantitative experiment of biofilm formation on nontranslucent biomaterial surfaces using the biofilm coverage rate (BCR). In this study, we quantified the time course of biofilm formation on a biomaterial (stainless steel) surface by means of BCR, viable cell count (VCC) with colony-forming units, and ATP-bioluminescence (ATP) as relative light units, and investigated the time-course relationship between biofilm development process and ica gene expression using reverse transcription-polymerase chain reaction (RT-PCR). S. epidermidis RP62A was inoculated on stainless steel washers and incubated for 0-8, 24, and 48 h. Biofilms attached to the washers were quantified by means of BCR, VCC, and ATP. RT-PCR of the ica gene was performed using total RNA isolated from biofilms at each incubation period. Results of these methods were compared. The amount of biofilms measured by BCR increased over time and particularly grew at 5-6 h into the incubation period. On the other hand, the results of VCC and ATP increased gradually, and at 24 h or 48 h the measurement values were very much greater than previously. Up to 8 h, there were significant correlations between BCR and VCC or ATP. The growth of BCR until 6 h is supported by RT-PCR of the ica gene. Compared with each result, two-dimensional biofilm occupation on a biomaterial surface is proposed to be rapidly completed within 6-8 h after bacterial attachment. Our data indicate that bacterial biofilms first grow two dimensionally with a producing matrix, and subsequently grow vertically and become mature.