The Relation between Oral Candida Load and Bacterial Microbiome Profiles in Dutch Older Adults Eefje A. Kraneveld1, Mark J. Buijs1, Marc J. Bonder1, Marjolein Visser2, Bart J. F. Keijser3, Wim Crielaard1, Egija Zaura1* 1 Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, Amsterdam, The Netherlands, 2 Department of Health Sciences, VU University Amsterdam, Amsterdam, The Netherlands, 3 Microbiology and Systems Biology, TNO Earth, Environmental and Life Sciences, Zeist, The Netherlands Abstract Currently there are no evidence-based ecological measures for prevention of overgrowth and subsequent infection by fungi in the oral cavity. The aim of this study was to increase our knowledge on fungal–bacterial ecological interactions. Salivary Candida abundance of 82 Dutch adults aged 58–80 years was established relative to the bacterial load by quantitative PCR analysis of the Internal Transcribed (ITS) region (Candida) and 16S rDNA gene (bacteria). The salivary microbiome was assessed using barcoded pyrosequencing of the bacterial hypervariable regions V5–V7 of 16S rDNA. Sequencing data was preprocessed by denoising and chimera removal, clustered in Operational Taxonomic Units (OTUs) and assigned to taxonomy. Both OTU-based (PCA, diversity statistics) and phylogeny-based analyses (UniFrac, PCoA) were performed. Saliva of Dutch older adults contained 0–4 6 108 CFU/mL Candida with a median Candida load of 0.06%. With increased Candida load the diversity of the salivary microbiome decreased significantly (p,0.001). Increase in the Candida load correlated positively with class Bacilli, and negatively with class Fusobacteria, Flavobacteria, and Bacteroidia. Microbiomes with high Candida load were less diverse and had a distinct microbial composition towards dominance by saccharolytic and acidogenic bacteria - streptococci. The control of the acidification of the oral environment may be a potential preventive measure for Candida outgrowth that should be evaluated in longitudinal clinical intervention trials. Citation: Kraneveld EA, Buijs MJ, Bonder MJ, Visser M, Keijser BJF, et al. (2012) The Relation between Oral Candida Load and Bacterial Microbiome Profiles in Dutch Older Adults. PLoS ONE 7(8): e42770. doi:10.1371/journal.pone.0042770 Editor: Joy Sturtevant, Louisiana State University, United States of America Received April 19, 2012; Accepted July 11, 2012; Published August 10, 2012 Copyright: 2012 Kraneveld et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The Longitudinal Aging Study Amsterdam is largely supported by a grant from the Netherlands Ministry of Health Welfare and Sports, Directorate of Long-Term Care. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have have read the journal’s policy and have the following conflicts: B.J.F. Keijser is employed by TNO Earth (Environment and Life Sciences), an organization that may benefit from a product or patent generated on the basis of the published data. In these cases, the authors will not receive additional salary, additional personal income, or any form of financial support. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors. * E-mail: e.zaura@acta.nl Introduction In the next decades healthcare services are faced with an aging population. In the European Union the proportion of 65 year and older is predicted to reach 53% of the total population by the year 2025 [1]. An aging population forms a health risk group due to several factors. First, there are internal factors such as senescence of tissues (e.g., mucosal fragility) and senescence of functions (e.g., lowered immune response). Secondly, there are extrinsic factors such as polypathologies (e.g., diabetes and malignancies), poly- medications and malnutrition [2]. The internal and external factors, which contribute to changes during aging, may also disturb the balance in the oral microbial ecosystem. A disturbance in the oral homeostasis between bacteria and fungi (e.g., Candida) may cause oral infectious disease. Candida species are a part of the commensal oral microbiota of healthy individuals at all ages with a reported prevalence between 15–75% [3,4] up to 80% in elderly, especially denture wearers [5,6]. The use of antibiotics and corticosteroids, chemotherapy, malnutrition, premature birth and old age are among the most common predisposing factors for opportunistic fungal infections i.e. mycoses [7]. The incidence of invasive fungal infections is on the rise. A study of the epidemiology of sepsis in the U.S. found that the annual number of cases of sepsis caused by fungal organisms increased by 207% between 1979 and 2000 [8]. Among the nosocomial bloodstream infections recorded in the U.S. between 1995 and 2002, 9.5% were fungal in origin with Candida spp. being the fourth leading cause of all cases [9]. Furthermore, the burden of invasive mycosis is shifting from the intensive care unit to the general hospital and even outpatient setting, where most types of mycosis represent endogenous infection in which the normally commensal host microbiota take advantage of the ‘‘opportunity’’ to cause infection [7]. Recent advances in research on bacterial–fungal inter-kingdom communication have shown that bacteria are capable of interfer- ing with or supporting the surrounding fungal community and vice versa [10,11,12]. It is expected that novel therapeutics will be based on ‘‘jamming’’ quorum sensing among pathogens and supporting communication between beneficial microbes. For practical reasons these studies are focusing on bacterial-fungal interactions in vitro at oversimplified conditions and with only a few species involved. No efforts so far have been made to assess the ecology and commensal relation of Candida with the oral microbiota at the breadth of the oral microbiome. There is an acute need to enhance our PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e42770