Oral Candida and Salivary Microbiome Figure 3. Salivary microbiome data by Candida load. Salivary microbiome data is plotted as OTU-based Principal Component Analysis (PCA) plot by Candida load in A) all subjects (N = 82) and in B) dentate subjects only (N = 34). Samples are colored by Candida load: green - low, blue - medium and red - high Candida load. Arrow indicates the main PC direction between low and high Candida load samples. doi:10.1371/journal.pone.0042770.g003 oral ecosystem due to other factors such as dry mouth or high carbohydrate intake which eventually may lead to increased Candida load) can only be elucidated in well-planned longitudinal studies. Our results bring us to two potential preventive strategies. First, the pH-lowering potential as well as duration of the low pH episode after carbohydrate consumption could be diminished by enrichment of base-producing bacteria. This could be achieved by modifying a diet from a carbohydrate- to an arginine-rich protein diet that could enhance the proportion of alkali-producing microbiota. Supplemen- tation with prebiotics such as arginine or urea [35,36] is also known to result in base production and counteracting acidification. So far clinical tests with these supplements have been focused on the inhibition of dental caries in a young and healthy population [37] and have not involved an older adult population at risk for Candida infection. Second, the finding of inverse relationship of certain taxa with the Candida load suggests a potential in search for and the use of natural, with oral health-associated probiotic strains, in fungi- bacteria warfare. These efforts will eventually bring us closer to developing strategies for prevention of microbial shifts and Candida overgrowth and to unraveling of and interfering with the interking- dom signaling processes. Conclusions There is a relation between oral salivary Candida load and the bacterial microbiome profile in older adults. Microbiomes with a high Candida load are less diverse and have a distinct microbial composition predominated by streptococci. The actual role of Candida as opportunistic pathogen in shaping the microbiome should be determined in further research including fungal- microbial interactions in complex microcosm models in vitro and following natural interactions in healthy and diseased populations during longitudinal clinical trials. Materials and Methods Subjects and Saliva Collection Study subjects (N = 82, mean age 66 years (SD 6.6); min 58, max 80 years; 50% females) were participants of the Longitudinal Aging Study Amsterdam (LASA) (http://www.lasa-vu.nl), a prospective study on predictors and consequences of changes in autonomy and well-being in the aging population in the Netherlands. The study was approved by the ethical review board of the VU University Medical Center (Amsterdam, the Netherlands), and all participants gave written informed consent. Subjects were chosen from three culturally distinct geographical areas in the West, Northeast, and South of the Netherlands. Details on the sampling and data collection procedures have been published elsewhere [38,39]. From the 2165 participants who participated in the measurement cycle in 2005–2006, 1421 participants were selected who were ,80 years of age, lived independently, had a good cognitive status (MMSE score.23 [40]) and were alive on January 15, 2007 for a sub-study on lifestyle including oral health. All selected persons received an extensive lifestyle questionnaire in February 2007 by mail. A total of 1058 participants (response 74.5%) returned a completed questionnaire (N = 326 no response, N = 18 refused, N = 8 not able due to physical or newly developed cognitive problems, N = 11 deceased). Informa- tion on oral health and dental status, gender and age was collected. Saliva was collected by the participants in the morning before they had taken breakfast, medicine, smoked tobacco, brushed their teeth or used any mouth rinse. Exclusion criterion was the use of antibiotics within the 3 months before saliva collection. Subjects were asked to remove their dentures during collection of saliva. Unstimulated saliva was collected by drooling into a DNA-free, sterile vial for 5 minutes [17]. The tubes were sent to the laboratory, saliva was alliquoted and stored at 280uC. PLOS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e42770