Real-time PCR results were not statistically different from the microarray results for each of the genes evaluated (p > 0.05). Figure 4 S. epidermidis transcriptome in mixed species biofilms and validation. Figure 4 A represents a heat map with hierarchal clustering of the samples. Red color indicates upregulation and light blue down regulation. S1, S2, S3 and SC1, SC2 and SC3 represent 3 biological replicates of single species S. epidermidis and mixed species biofilms respectively. Two down
regulated genes (lrgA and lrgB) and 3 upregulated genes (prfA, hrcA and guaC) were evaluated for microarray validation (Figure 4 B). Results for microarray are shown in white bars and real-time RT PCR in gray bars. Real-time RT PCR shows consistent results with microarray (p > 0.05 for each gene tested). Evidence for increased eDNA in mixed-species biofilms Quantification selleck products of the bacterial eDNA in the extracted TGF-beta/Smad inhibitor biofilm matrix using S. epidermidis specific primers (lrgA, lrgB and bap) showed significantly increased bacterial eDNA in mixed-species biofilms of S. epidermidis and C. albicans compared to single
species biofilms KU-57788 mw of S. epidermidis (Figure 5A). Extracted biofilm eDNA was normalized for CFU/ml of the initial organism suspension used to form the biofilms. In order to understand the contribution of eDNA from Candida, we assayed the eDNA with Candida chromosomal gene specific primers RIP, RPP2B and PMA1 (Figure 5B). Candida specific eDNA was identified in single species Candida biofilms
(< 30 ng/108 CFU/ml), none in S. epidermidis single species biofilms and negligible in mixed species biofilms. This confirms the predominance of bacterial (Staphylococcal eDNA) in the extracellular matrix of mixed-species biofilms. Figure 5 Increased eDNA in the mixed-species biofilms confirmed by real-time RT PCR. Biofilm matrix was extracted and eDNA was quantitated by real-time RT PCR using genomic DNA as standard. Primers for S. epidermidis genes (lrg A, lrgB and bap) were used to quantify the eDNA (Figure 5 A). Staphylococcal eDNA was increased significantly in the mixed species biofilms compared to single species S. epidermidis biofilms (*, ** and ¶, p < 0.05). DNA Synthesis inhibitor Candida gene specific primers (RIP, RPP2B and PMA1) were used to assess the contribution of eDNA by Candida in mixed species biofilms (Figure 5 B). Candida specific eDNA was present in Candida biofilms, absent in S. epidermidis biofilms and negligible in mixed species biofilms. S. epidermidis biofilms are represented in white bars, mixed species biofilms in gray bars and Candida biofilms in chequered bars. Disrupting eDNA by DNAse decreases single and mixed-species biofilms We further confirmed the presence of eDNA by estimating the effects of DNA degradation on single and mixed species biofilms. DNAse I treatment for 16 hrs disrupted both single and mixed species biofilms of S.