Oral Candida and Salivary Microbiome Figure 1. Relative abundance of bacterial taxa in saliva samples at the class level. The sample order corresponds to the increase in Candida load in the samples (Table S2). The Candida load was measured as proportion of ITS gene over 16S gene abundance by qPCR. Increase in Candida load correlated positively with reads classified as Bacilli (p = 0.013; Spearman’s rho 0.274), while class Bacteroidia (p = 0.001; Spearman’s rho 20.360), Flavobacteria (p = 0.003; Spearman’s rho 20.322), Fusobacteria (p = 0.012; Spearman’s rho 20.275) correlated negatively with the Candida load (Figure S1). doi:10.1371/journal.pone.0042770.g001 Individual Microbiomes of the Dentate Group by Candida Load To exclude the covariance of dentures on the relation of the Candida load with the microbiome profiles, we used the dentate group (N = 34) alone. The diversity statistics (Figure 4) showed that among the dentate subjects, as with the entire study group, the samples with a high Candida load (N = 14) were less diverse (Shannon Diversity Index 2.3, SD 0.4) than samples with low Candida (N = 8) (mean 2.7, SD 0.2) (p = 0.005, Mann-Whitney test). Additionally, the Dominance Index was significantly higher in the high Candida load samples (p = 0.012). The PC 1 (18% of variance) and PC 2 (13% of variance) of the OTU-based PCA positioned most of the low Candida load samples and some medium load samples in the upper right quadrant of the PCA distant from the high Candida load samples (Figure 3B). The PCoA of the unweighted UniFrac distances showed that samples with a low Candida load were less dispersed than medium and high Candida load samples (Figure S3C). Weighted UniFrac (Figure S3D) separated low and high Candida samples in a similar way to the PCA (Figure 3B). Loadings of the PCA showed that genus Streptococcus (4 OTUs) contributed to samples from the high Candida load, while Prevotella (3 OTUs), Leptotrichia (2 OTUs), Neisseria and Porphyromonas determined the position of the low Candida load samples. The low Candida load was associated with higher abundance of OTUs classified as Prevotella, Porphyromonas and Haemophilus (p,0.05; ANOVA; NS after Bonferroni correction). Discussion In this study we assessed the relation between the Candida load and the bacterial profiles of saliva of Dutch older adults. With increased Candida load the diversity of the salivary microbiome decreased and the composition changed towards dominance by Bacilli (streptococci and lactobacilli) and disappearance of genera within class Fusobacteria and Bacteroidia. The microbiome of dentate subjects differed from that of the fully edentate, but this difference was not significantly associated with the Candida load. Saliva samples of our study group showed a large variation in Candida load: 0–46108 CFU/ml with a median Candida load of 0.06%. Studies on comparable population report about 0.02% Candida in saliva, detected by culturing [5,13,14]. All but two (97%) of our samples were Candida-ITS gene positive. Studies on a similar age group, though using a culturing approach, have reported around 80% Candida prevalence [5,6]. This difference could be due to the lower detection limit and higher selectivity of the culturing technique compared with the qPCR on Candida- specific internal transcriber spacer (ITS) region, used here. Due to the study logistics it was not possible to collect samples from defined intraoral habitats such as plaque, tongue or denture surfaces. Although different intraoral niches select for a habitat-specific microbiome and salivary microbial profiles show higher similarity to mucosal sites than to dental surfaces [15], saliva is frequently preferred as a representative oral sample for large clinical studies [16]. Dentures, especially in edentate individuals are known to be one of the main predisposing factors for outgrowth of Candida frequently leading to denture stomatitis [17]. In our study population we found only a trend for higher Candida load in edentulous full prosthesis wearers. The lack of statistical significance could be explained by the small sample size (20 edentulous individuals) and thus lack of statistical power, as well as by good oral and general health of the study participants. It has been shown that denture stomatitis is a multifactorial disease where poorly fitting dentures, continuously worn dentures, poor oral hygiene and reduced salivary flow all increase the ability of Candida to colonize the dentures and contribute to the etiology of the disease [14,17]. Saliva profiles of Dutch older adults were composed of commensal bacteria commonly found in human salivary micro- biomes [15,16,18]. Pathogenic species, such as enterobacteria, PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e42770