Relative reverse-transcription real-time PCR and microscopy for studying slime production by Staphylococcus epidermidis under various physicochemical and flow conditions
Abstract number: O209
Foka A., Katsikogianni M., Chini V., Missirlis Y.F., Anastassiou E.D., Spiliopoulou I.
Objectives:Staphylococcus epidermidis has emerged as a pathogen associated with infections of implanted medical devices impeding their long-term use. Characteristics of S. epidermidis that allow persistence of infection are the ability of bacteria to adhere to surfaces in multilayered cell clusters, followed by the production of a mucoid substance more commonly known as slime, encoded by the ica operon. The adherent bacteria and slime are collectively known as biofilm. The coupled effects of specific chemical terminal surface groups and flow conditions on slime production and biofilm formation by S. epidermidis were investigated in correlation to the expression of two genes of the ica operon.
Methods: Reference control strains (ATCC35984, slime-positive and ATCC12228, slime-negative), and two clinical strains isolated from different hospitalised patients, (one ica-positive/slime-positive and one ica-positive/slime-negative) were tested. Bacteria grown in BHI medium were suspended in physiological saline at a concentration of ~3×109 cells/ml. Hydroxyl (OH)-terminated (hydrophilic) and methyl (CH3)-terminated (hydrophobic) glass surfaces were used as substrates in a parallel plate flow chamber. Bacterial adhesion was examined under two flow rates: 2 ml/min and 20 ml/min for two and four hours. Total RNA from both planktonic (p) and adherent (a) bacteria, after detachment with trypsin, was isolated by the Trizol method. Reverse transcription followed by relative Real-Time PCR (rRT-PCR) towards a 207 bp part of 23S rRNA gene, allowed the detection of expression levels of icaA and icaD. Adherent bacteria were investigated with Scanning Electron and Confocal Laser microscopes.
Results: Higher expression levels of both icaA and icaD genes onto glass and especially methyl-terminated glass surfaces were calculated by rRT-PCR, under higher flow rate in two hours by the reference and the clinical slime-positive strains. These results correlate well with adherent bacterial cell counts and images taken by both microscopes. The ica-positive slime-negative clinical strain showed lower expression levels of ica genes, less adherent ability and PIA production on glass surfaces, as observed by microscopes.
Conclusions: Higher flow rate enhances the expression level of both ica genes, with a peak in two hours. Hydrophobic biomaterial surfaces seem to play a crucial role to initial adherence, increasing ica gene expression and PIA synthesis.
|Session name:||19th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Helsinki, Finland, 16 - 19 May 2009|
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