Figure 2. Diversity statistics of salivary microbiomes by Candida load. Diversity statistics of salivary microbiomes as: A) Dominance Index and B) Shannon Diversity Index by log10 Candida ITS/16S gene. Candida load correlated positively with the Dominance Index (p,0.001; Spearman’s rho 0.388) and negatively with the Shannon Diversity Index (p,0.001; Spearman’s rho 20.403). doi:10.1371/journal.pone.0042770.g002 staphylococci and pseudomonas were found only in few subjects. Our study group was not representative of a Dutch elderly population, but consisted of non-institutionalized, self-supportive older adults with a good general health, which might explain the low prevalence of the nosocomial i.e. hospital-acquired pathogens. However, in two cases relatively high proportions of pathogenic species were found: staphylococci in a single sample (LASA161) contributed to 9% of the reads, and two Pseudomonadacea OTUs contributed to 11% of the reads in an another sample (LASA137). Interestingly, both of these cases belonged to high Candida load group. Candida has been previously co-associated with Staphylococcus aureus and Pseudomonas in human infections [11]. It has been shown that C. albicans physically interacts with S. aureus and differentially regulates specific virulence factors of S. aureus in vitro [19]. We observed a negative correlation between the Candida load and the diversity of the salivary microbiome. We also showed that absence of teeth and presence of full dentures as such already were related to decreased microbiome diversity, irrespective of the Candida load. Therefore the effects of Candida load were assessed also on the dentate subgroup. Decreased diversity was seen both in the entire study sample and in the subgroup of dentate subjects. Decreased bacterial diversity is associated with a disbalanced community, e.g., the salivary microbiome from caries-active individuals was shown to be less diverse than microbiome from caries-free individuals [20]. Perturbations such as prolonged low pH episodes reduce the diversity of microbial communities due to Oral Candida and Salivary Microbiome acidification of the environment [21]. The importance of equal sample size on accuracy of diversity analyses was recently emphasized [22]. To avoid the bias of under- or oversampling, we used randomly subsampled dataset to the lowest number of reads per sample for all analyses. The proportion of certain bacterial classes such as Fusobacteria, Bacteroidia, and Flavobacteria correlated negatively with the Candida load. These taxa are generally less acid-tolerant than Bacilli, which was the only class that increased with the increasing Candida load. This suggests that the oral environment of a high Candida load samples was acidified, i.e. exposed to prolonged low pH episodes. Oral Candida isolates have been shown to be highly acid tolerant and acidogenic [23] and able to lower the pH of a culture during growth from pH 7.5 to pH 3.2 [24]. Within the class Bacilli, streptococci increased most with increased Candida load. The interactions between fungi and streptococci appear to be synergistic [25,26]. The growth of Candida albicans is enhanced if co-cultured with oral streptococci in the presence of sucrose [27]. Streptococcus sanguinis, Streptococcus gordonii, Streptococcus oralis and Streptococcus anginosus are known to coaggregate with C. albicans, especially if the yeast cells are subjected to glucose starvation [26]. These streptococcal species are able to adsorb protein components from human saliva (basic, proline-rich proteins) that in turn promote C. albicans adhesion [28]. The adherence of S. gordonii is mediated through streptococ- cal cell surface polysaccharide receptors and polypeptide adhesins [29]. Candida, on the other hand, in addition to reducing the oxygen tension to levels preferred by streptococci, may provide growth stimulatory factors for the bacteria as a result of nutrient metabolism [11]. Next to streptococci, also lactobacilli were associated with high Candida load. While most lactobacilli are recognized as probiotics regarding Candida control [30], Lactoba- cillus casei was shown to stimulate germ tube growth – an important C. albicans virulence factor [30,31]. Both, streptococci and lactobacilli are in general aciduric and acid tolerant microorgan- isms, often associated with dental caries pathogenesis [32]. A low Candida load was associated with among others, genus Prevotella, Porphyromonas and Peptostreptococcus. There are no compara- ble clinical reports regarding any relationships of these bacteria with fungi. In in vitro studies however, two putative periodontal pathogens – Prevotella nigrescens and Porphyromonas gingivalis – have been shown to exert a suppressive effect on C. albicans viability [33], while P. gingivalis and Prevotella intermedia inhibited C. albicans germ tube formation in vitro [31]. With respect to the complexity of the oral ecosystem, it is not likely that each of the taxa is interacting with Candida or is affected by it directly. Our finding of Peptostreptococcus together with Prevotella in low Candida load samples might be due to a symbiotic relationship between these two taxa. For instance, it has been shown that Peptostreptococcus anaerobius is nutritionally dependant from Prevotella bovia (two species involved in pathogenesis of bacterial vaginosis), where P. bovia provides amino acids required for the growth of P. anaerobius [34]. Most probably multiple intermediate interactions among different metabolically dependent bacterial taxa as well as environmental conditions are affected by Candida or are affecting the presence of Candida. The exact mechanisms should be investigated further, both in longitudinal clinical trials and in fundamental experimental research in vitro. The current findings strongly suggest that acidification of the environment coincides with the high load of Candida and is the major ecological factor that perturbs the oral commensal microbiome leading to the shift towards an increase of aciduric microbiota and reduction of natural diversity of the bacterial microbiome. The actual sequential order of the events (i.e., what comes first – Candida outgrowth followed by acidification or primary acidification of the PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e42770