Signal intensity values
were extracted from scanned images using GenePix® Pro 6 software (Molecular Devices). The raw gpr files were loaded in Genespring GX 11.5, the data log2 transformed; background corrected, and normalized using Ferrostatin-1 the Quantile algorithm. Hierarchical clustering map was generating using Euclidean algorithm with the average linkage rule. Differential gene expression between the two samples groups (S. epidermidis and mixed species biofilms) was evaluated by unsupervised unpaired t-test on the log2 BAY 11-7082 clinical trial transformed mean data. A fold-change ratio (mixed species biofilms vs. S. epidermidis biofilms) was calculated with a fold change cutoff of 1.5 and p-value of 0.05. Probe set lists were trimmed to represent S. epidermidis and analyzed using unpaired t-test and Benjamini-Hochber multiple-testing correction to generate check details targeted lists of differential expression. Microarray expression patterns were validated using real-time PCR using three upregulated and
two down regulated genes. Quantitation of eDNA in single and mixed-species biofilms Biofilm matrix and eDNA were extracted from 24 hr single species S. epidermidis biofilms and mixed species biofilms of S. epidermidis and C. albicans as described previously [30, 39, 46]. The extracellular matrix from harvested biofilms was carefully extracted without cell lysis and contamination with genomic DNA as described [30, 39, 46]. The amount of eDNA was quantified by real-time RG7420 price RT-PCR using standard curves of known quantities of S. epidermidis and C. albicans genomic DNA. Real-time PCR was performed using the SYBR Green kit (Qiagen) and primers for 3 chromosomal genes of S. epidermidis, lrgA, lrgB and bap (whose primers for RT-PCR were previously optimized in our lab) or stably expressed chromosomal genes of C. albicans RIP, RPP2B and PMA1[49]. The amount of measured eDNA was normalized for 108 CFU organisms in the initial inoculation. Effects of DNAse on single and mixed species biofilms Concentration dependent effects of DNAse I (Sigma or Roche, USA) was studied by exposing 24 hr single and mixed-species biofilms, at 0 to 1.25 mg/ml concentrations DNAse I for
16 hr and residual biofilm evaluated by measuring absorbance at 490 nm after XTT reduction [50]. A time course experiment was performed by the addition of DNAse (0.65 mg/ml) at 0, 6 or 18 hrs of biofilm development. The biofilms were developed for a total of 24 hr and metabolic activity quantitated by XTT method and measuring absorbance at 490 nm. Percentage reduction in biofilms compared to controls was evaluated for single and mixed species biofilms at DNAse exposures starting at 0, 6 or 18 hrs. Data deposition The microarray dataset supporting the results of this article has been deposited and available at the NCBI gene expression and hybridization data repository (http://www.ncbi.nlm.nih.gov/geo/), [GEO accession number GSE35438].