Quantitative analysis of Staphylococcus epidermidis biofilm on the surface of biomaterial.

Background: 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).

Methods: 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.

Results: 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.

Conclusions: 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.