Figure 4. Diversity statistics by Candida load in dentate subjects. Diversity statistics by Candida load (low (N = 8), medium (N = 12), high (N = 14)) in dentate subjects as boxplots of: A) OTUs per sample, B) Dominance Index and C) Shannon Diversity Index. Each box shows the median, quartiles, and outliers (circles). Connector connects statistically significantly different groups (p,0.05; Mann-Whitney test). doi:10.1371/journal.pone.0042770.g004 DNA Extraction Salivary DNA was extracted as described previously [16]. In brief, 0.1 ml of samples was lysed in phenol in a bead beating procedure, using 0.25–0.5 mm diameter (for breaking up the yeast cells) and 0.1-mm diameter (for breaking up the bacterial cells) Oral Candida and Salivary Microbiome glass beads (Lab Services BV, the Netherlands). DNA was extracted with the AGOWA mag Mini DNA Isolation Kit (AGOWA, Berlin, Germany) and samples were stored at 220uC until further analysis. qPCR Real-time qPCR was used to determine the concentration (CFU/ml) of Candida and bacteria in the saliva samples. Primers specific for the Candida ITS rDNA gene (Forward: CCTGTTTGAGCGTCRTTT; Reverse: TTCTCCGCTTATTGATAT) modified from [41] were used to determine the Candida concentration. Primers and probe specific for the prokaryotic 16S rDNA gene (Forward: TCCTACGG- GAGGCAGCAGT; Reverse: GGACTACCAGGGTATCTAATCCTGTT, Probe: 6FAM-CGTATTACCGCGGCTGCTGGCAC-BBQ) [42] were used to determine the bacterial concentration. Real-time qPCR was performed using a LC480-II light cycler (Roche Diagnostics, Switzerland). As internal control for PCR inhibition a qPCR on PhHV (Phocid herpesvirus type 1 gB gene) was performed in all reactions and DNA and primers where added according to Watzinger et al. (2004) [43]. Reactions were performed with 3 ml of DNA. The total reaction volume was 20 ml. Reactions contained a 26 PCR Probe Master Mix (Roche) or, in the case of ITS primers, a 26 SYBR Green PCR Master Mix (Roche). For 16S, 7.5 pmol primers and 3.8 pmol probe, for PhHV 1.8 pmol primers and 0.4 pmol probe and for ITS 24 pmol of each primer was used. The conditions for the qPCR reaction were: an activation step of 10 min at 95uC followed by 50 cycles consisting of a denaturation step at 95uC for 30 sec, an annealing step at 60uC for 30 sec and an extension step at 72uC for 30 sec. Standard curves obtained from the overnight cultures of Candida dubliniensis and Escherichia coli were used to extrapolate DNA concentrations (CFU/ml) of the Candida (ITS) and bacterial (16S) concentration (CFU/ml) in the saliva samples, respectively. The CFU numbers were validated in saliva samples spiked with six different Candida overnight cultures (Candida tropicalis, Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei and Candida kefyr) and performing both, culturing of Candida and performing Candida specific qPCR. Amplicon Preparation and Pyrosequencing Amplicon libraries of the small subunit ribosomal RNA gene V5–V7 hypervariable region were generated for each of the individual samples. PCR was performed using the forward primer 785F (GGATTAGATACCCBRGTAGTC) and the reverse primer 1175R (ACGTCRTCCCCDCCTTCCTC). The primers included the 454 Life Sciences (Branford, CT, USA) Adapter A (for forward primers) and B (for reverse primers) fused to the 59 end of the 16S rDNA bacterial primer sequence and a unique 10 nt sample identification key. The amplification mix contained 2 units of Pfu Ultra II Fusion HS DNA polymerase (Stratagene, CA, USA), 1 unit Buffer Pfu Ultra II [10x], including 2.0 mM MgCl2 (Stratagene), 240 mM dNTP (Fermentas GMBH, Germany) 0.5 mM of each primer. To each reaction 100 pg of DNA template was added. After denaturation (95uC; 2 min), 9 cycles were performed consisting of denaturation (95uC; 30 sec), annealing (53uC; 30 sec), and extension (72uC; 80 sec), followed by 23 cycles of denaturation (95uC; 30 sec), annealing (62uC; 30 sec), and extension (72uC; 80 sec). The amplicons were purified by means of the IllustraTM GFXTM PCR DNA and Gel Band Purification Kit (GE Healthcare, Eindhoven, the Netherlands). The quality and the size of the amplicons were analyzed on the Agilent 2100 PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e42770